Methods of treatment using pluripotent human adipose adult stem cells

ABSTRACT

Methods for the efficient isolation and use of pluripotent adipose-derived stem cells (PASCs) are provided. In certain embodiments the methods involve providing an adipose tissue sample from which the stromal vascular fraction is co-cultured with the adipocyte fraction. PASCs can be isolated with a high degree of purification without requiring an additional cell enrichment process (e.g. cell sorting). PASCs and their conditioned media can be used for tissue regeneration within hours of harvesting the adipose tissue, and without requiring cell expansion. PASCs can grow as floating individual cells, as clusters of cells, or attached to surface(s) of the culture vessel. PASCs do not produce teratomas in vivo, nor do they induce immunorejection upon transplantation, and they achieve a high efficiency in grafting. The cells and compositions can be used for cell therapy and to screen new drugs.

This application is a continuation of U.S. patent application Ser. No.16/179,798, filed Nov. 2, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/893,014, filed Nov. 20, 2015, which is anational stage of PCT/US14/39137, filed May 22, 2014, which applicationclaims priority to U.S. provisional patent application No. 61/826,417,filed May 22, 2013, the entire contents of which are incorporated hereinby reference.

BACKGROUND

Pluripotent stem cells have the ability to differentiate into all typesof cells (endodermal, ectodermal and mesodermal origin) and thereforehave the potential to regenerate any kind of tissue with propermanipulation. Currently, two gold standard pluripotent stem cells areknown, embryonic stem cells (ESCs) and induced pluripotent stem cells,which are reprogrammed adult somatic cells (iPSCs).

ES cells have unequivocally taken center stage in the field of stem cellresearch. ES cells exhibit the potential to treat a plethora ofpreviously irreversible disorders through their capacity to generatetissues and thus to revolutionize regenerative medicine (1-3). However,evidence has since emerged that ES cells exhibit high rates ofimmunorejection upon transplantation and form teratomas as a result oftheir unbridled proliferation (4). In conjunction with debatessurrounding the bioethical issues concerning the usage of human embryos,this teratogenic propensity precludes the practical application of EScells in regenerative medicine.

Addressing the ethical dilemmas surrounding the use of ES cells for celltherapy, iPS cells became of interest in the stem cell field (5-6). iPScells have the capacity to re-program, through an intricate mechanisminvolving the induction of the so-called “Yamanaka factors,” includingNanog, Oct 3/4, Sox2, c-Myc and Klf4, which subsequently became thecharacteristic markers that establish pluripotency: the ability toself-renew and generate cells from the three germ lines and thus formteratomas (7-9). Though iPS cells resolve concerns of immunorejectionbecause they can be generated from a patient's own, or autologous,cells, as well as the ethical issues that hinder the use of stem cellsextracted from human embryos, the production of teratomas upontransplantation as a result of unbridled cell proliferation andextremely low survival rate of both iPS and ES cells upon reintroductionto the host organism, impede the translational use of these cells(10-12). Furthermore, it has also been found that mature iPS cellspossess an epigenetic memory, defined by the remnants ofposttranslational histone and DNA modifications, preventative ofentirely successful reprogramming, often restricting their physiologicalfunction to that of a cell within the same lineage as the original stemcell source (13-15). Investigators have made attempts to address theseissues, but to little avail (16-17). Despite excessive monetary andtemporal efforts devoted to the study of both ES cells and iPS cells,there has been little progress made in overcoming the hurdles facingthese stem cells and their use for cell therapy.

Other, non-reprogrammed pluripotent stem cell populations have caughtthe attention of the scientific community as an alternative to ethicallycontentious ES cells and genetically modified iPS cells. However, thoughmultiple populations of adult stem cells have been put forth, many havefaced a great deal of suspicion due to irreproducibility. Isolated frombone marrow, multipotent adult progenitor cells (MAPCs), bothpluripotent and non-tumorigenic, were reported to contribute to chimericoffspring when injected into a mouse model and to regenerate damagedtissue in vivo (18-19). Human marrow-isolated adult multilineageinducible (MIAMI) cells and very small embryonic-like stem cells(VSELs), isolated from umbilical cord blood in addition to bone marrow,were soon to follow, exhibiting similar pluripotent and non-tumorigenicproperties. Like VSELs, unrestricted somatic stem cells (USSCs),isolated from umbilical cord blood, are reportedly pluripotent but lackthe classic pluripotent stem cell marker expression (20). These adultpluripotent stem cell lines have all been publically flagged for furtherinvestigation and reproduction, or in the case of VSELs, negatedentirely. Stimulus-triggered acquisition of pluripotency (STAP),characterized by exposing splenic CD45+ lymphocytes to acidic conditionsfollowed by incubation with leukaemia inhibitory factor (LIF), hasrecently been described as a method of bestowing pluripotency uponsomatic cells (21). However, STAP cells form teratomas, hindering theirclinical application. STAP cells are currently under investigation todetermine the overall validity of the published results as well as themechanism behind their reprogramming.

A population of human pluripotent stem cells with a highpost-transplantation survival rate that does not undergo teratogenesisin vivo can facilitate treatment of many disorders affecting humanbeings. Recently, a group of researchers from Tohoku University, Japan,isolated and cultured a stem cell population isolated from skin and bonemarrow with pluripotent characteristics (22-23). These cells named Musecells (Differentiating Stress-Enduring Multilineage Cells) can becreated in vitro under cellular stress conditions. Muse cells grow whileforming cell clusters. Muse cells have been characterized as mesenchymalstem cells that have the ability to express a set of genes associatedwith pluripotency.

Furthermore, Muse cells can differentiate into endodermal, ectodermal,and mesodermal cells both in vitro and in vivo. More importantly, unlikeESCs and iPSCs, when Muse cells are transplanted (by local or i.v.injection) into immunodeficient mouse models for tissue regeneration,Muse cells integrate into damaged skin, muscle, or liver and regeneratenew tissue. Muse cells do not undergo tumorigenic proliferation, andtherefore would not be prone to produce teratomas in vivo, nor do induceimmuno-rejection in the host upon autologous transplantation (22-23).Furthermore, in contrast to iPSCs, Muse cells do not requireintroduction of exogenous genes for their pluripotency. In addition,Muse cells are shown to home into the damage site in vivo andspontaneously differentiate into tissue specific cells according to themicroenvironment to contribute to tissue regeneration when infused intoblood stream (22).

Muse cells have the potential to make critical contributions to tissueregeneration, but are hindered due to difficulties associated withextraction of bone marrow stromal cells and human skin fibroblasts, andtime-consuming purification methods including cell sorting, cell cloningby limiting dilution, long periods of cell culture which lead to a finalproduction of only 1,000,000 Muse cells from of bone marrow stromalcells or human skin fibroblasts after SSEA3 cell sorting and one monthof cell expansion. There thus remains a need for isolation ofnon-tumorigenic pluripotent cells as a source of tissue regeneration,and for improved methods of isolating such cells.

SUMMARY

Advances in stem cell therapy face major clinical limitations,particularly challenged by autologous transplantation of humanpluripotent stem cells that does not undergo teratogenesis in vivo witha high post-transplantation survival rate. Hostile host factors of theengraftment microenvironment such as hypoxia, nutrition deprivation,pro-inflammatory cytokines, and reactive oxygen species can eachcontribute to unwanted differentiation or apoptosis. The inventionaddresses these needs and others by providing cells, methods andcompositions for ameliorating tissue damage and disease, as well as foruse in identifying, screening and testing new therapeutic agents.

In one embodiment, the invention provides a method of isolatingpluripotent adipose stem cells (PASCs) from adipose tissue. The methodtypically comprises the steps of: (a) providing an adipose tissuesample; (b) subjecting cells in said sample to stress conditions; (c)co-culturing adipocytes and a stromal vascular fraction for 2-36 hours,typically for 6-8 hours; (d) recovering the viable cells; and (e)optionally culturing the recovered cells. In one embodiment, the stressconditions of step (b) comprise incubating the cells in a mediumcontaining a proteolytic enzyme. The co-culturing of step (c) can beperformed in the presence of a proteolytic enzyme as well. In a morespecific embodiment, the enzyme is collagenase.

The stress conditions of step (b) include, but are not limited to,protease treatment, exposing cells to starvation conditions (nonutrients, no glucose), hypoxic conditions (lack of oxygen), lowtemperatures, heat shock, and lysis by mechanical procedures such assonication. Two or more such stressors may be used together. Under theseconditions, a highly purified population of PASCs is isolated withoutthe need for time-consuming cell sorting methods, magnetic beads orspecial devices and prolonged cell culture procedures. The stressconditions are typically applied through the co-culturing step. Forexample, the cells obtained from adipose tissue are treated initiallywith protease (e.g., collagenase) for about 45 minutes at 37° C.,followed by the stress conditions of step (b), in which the cells areexposed to hypoxic conditions at 4° C. for 2-8 hours (or overnight; step(c)), during which time the proteolytic activity is permitted tocontinue.

In a typical embodiment, the co-culturing is performed in the absence ofserum. For example, the co-culturing can be performed in Dulbecco'sMinimum Essential Medium (DMEM), or a similar basic medium known in theart. The co-culturing can be as simple as storing the cells in a 50 mlcentrifuge tube. Stressors can be applied, for example, by closing thecap of the tube to create hypoxic conditions, placing the tube or othercontainer into a refrigerator (cooling), as well as by using anutrient-free medium (starvation).

In one embodiment, the co-culturing is performed for 4-24 hours.Typically, the co-culturing is performed for 6-8 hours (or overnight).The length of the co-culturing can be modified to accommodate timingconsiderations. For example, if isolated PASCs are required on an urgentbasis, such as for treatment of traumatic injury, steps (b) and (c) canbe shortened, and multiple stressors applied, to accelerate the processof selecting for stress-resistant cells.

In one embodiment, the recovering of step (d) comprises recovering atleast 200,000 PASCs/ml of lipoaspirate material. This is equivalent to200,000,000 PASCs retrieved with one liter of lipoaspirate material. Inother embodiments, the recovering comprises recovering at least50,000,000 PASCs, or at least 100,000,000 PASCs/lt of lipoaspiratematerial. These quantities of PASCs are typically recoverable within 6-8hours (or overnight) of initiating the stress conditions of step (b). Insome embodiments, at least 500,000,000 PASCs/liter of lipoaspirate arerecovered within 8 hours of initiating the stress conditions of step(b). In other embodiments, the PASCs are recovered within 12-16 hours.In one embodiment, the method is performed without cell-sorting. In oneembodiment, the recovering of viable cells comprises centrifuging theculture medium, removing the supernatant, washing the remaining cellpellet and resuspending the cell pellet in a buffer solution and/orculture medium. In some embodiments, the recovering of viable cellscomprises providing red cell lysis buffer in the solution or culturemedium containing the cells.

The invention further provides PASCs as described herein. The PASCs aretypically present in a composition that is essentially free of othercell types (e.g., at least 95% pure, and in some embodiments, at least99% pure). Also provided is a composition comprising PASCs isolatedaccording to the method described herein, or progeny thereof, and atherapeutically acceptable culture medium. Also provided is acomposition comprising conditioned medium that has been recovered from aculture of PASCs isolated in accordance with the methods describedherein. Culture conditioned medium contains factors (cytokines,chemokines, growth factors, peptides, proteins) secreted by PASCs keptin culture for 24-72 hours. The conditioned medium is typically preparedfrom PASCs that have been cultured under standard conditions (e.g. DMEM,10% fetal calf serum at 37C, 5% CO2). These factors released into theconditioned medium of PASCs have anti-inflammatory and/orimmunomodulatory properties that can be used for treatment of disease,particularly immunological or autoimmune diseases.

In one embodiment, the composition comprises PASCs isolated fromlipoaspiration. In one embodiment, the lipoaspiration is performed onthe subject to whom the composition is administered. Both autologous andallogeneic transplantation of PASCs are contemplated. In a typicalembodiment, the composition is administered to the subject within 6-24hours of the lipoaspiration or less.

The invention additionally provides a method of ameliorating tissuedamage in a subject. Typically, the method comprises administering acomposition of the invention to the subject. Where the compositioncomprises PASCs, it may be administered under conditions permitting thePASCs of the composition to divide and to populate a site of tissuedamage, although migration of PASCs to the site of damage is notrequired in all embodiments.

In some embodiments, the tissue damage comprises traumatic injury ordisease-associated damage. Representative examples of traumatic injurycomprises hypoxia, bone injury, laceration, and gunshot wound. Inanother embodiment, the disease-associated damage comprises damageassociated with diabetes, vascular disease, infection, degenerativeneurological disease, cancer, Huntington's Disease, Multiple Sclerosis,Rheumatoid Arthritis, Lupus, Diabetes type I, Crohn's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrates PASCs in (1A) adipose tissue which also containsadipocytes, the stromal vascular fraction including adipose tissuemacrophages (ATMs) and multipotent adipose stem cells (ADSCs) amongother cell types, (1B) adipocyte fraction (floating cells)(photomicrograph) and in (1C) the stromal vascular fraction(photomicrograph image), PASCs are present in both fractions (markedwith circles).

FIG. 2 illustrates the high degree of cell plasticity in the differentcell components of adipose tissue. ADSCs can differentiate alongadipocyte, osteoblast, chondrocyte, and other mesenchymal cell lineagesin a manner similar to that of multipotent stromal cells derived frombone marrow. It is generally accepted that mature adipocytes do notregularly undergo mitosis, and thus, an increase in adipocytes usuallyreflects a differentiation of preadipocytes. However, several studiesindicate that mature adipocytes could also have proliferative activity.Recent studies suggest adipocytes can dedifferentiate to preadipocytesand can even differentiate to a multipotent cell population. Of note,preadipocytes have also been observed to rapidly and efficientlydifferentiate into typical macrophages and vice-versa demonstratingsignificant plasticity of these cells.

FIG. 3 illustrates another example of cell plasticity present in adiposetissue indicated by the capacity of adipose tissue macrophages(hematopoietic stem cell origin, CD14 (+) cells) to be differentiated topreadipocytes (mesenchymal stem cell origin, S-100 (+) cells). DuringATMs differentiation to preadipocytes, cells express both CD14 (+) andS-100 (+) markers. Most preadipocytes observed were CD14 (+) to varyingdegrees. As macrophages differentiated towards a preadipocytemorphology, the cells expressed less CD14 and more DLK/S-100.

FIG. 4 illustrates the presence of pluripotent adipose stem cells(PASCs) in adipose tissue. After collagenase treatment andcentrifugation, the adipocyte fraction (floating cells) and the stromalvascular fraction (SVF) (cell pellet) are released from adipose tissue.The digested material is then subjected to severe cellular stress whichincludes long incubation with collagenase (6-8 hrs), lack of nutrients,low temperature, high hypoxia and also the paracrine/autocrineinteraction between the adipocyte fraction and the stromal vascularfraction, which lead to the release of cytokines/chemokines fromadipocytes and cells present in the SVF. PASCs are the only cell typethat can survive such external stress conditions. PASCs are present inboth the adipocyte and stromal vascular fractions.

FIG. 5 illustrates a simple scheme of PASCs isolation from lipoaspiratematerial. After 30 min/37° C. digestion of adipose tissue, all cellsreleased from adipose tissue were exposed to severe cellular stress(extended incubation with collagenase, lack of nutrients, lowtemperature, high hypoxia, release of cytokines/chemokines fromadipocytes and other cell components). PASCs are the only cell type thatcan survive such external stress. PASCs are present in both adipocyteand stromal vascular fractions. PASCs can grow as individual cells andalso by forming clusters of cells in suspension, as well as growing asadherent cells. Based on their pluripotent characteristics, non-teratomaformation and high percentage of grafting, PASCs have the potential tobe used use in tissue engineering, reconstructive medicine, andtreatment of degenerative and immune disorders

FIG. 6 is a schematic representation of the different proceduresincluding liposuction, collagen digestion of the aspirated fat, PASCsisolation and injection of PASCs (via i.v. or into the damage tissue).This procedure is extremely fast between (5-7 hours) and efficient(1,000,000 PASCs/1 of lipoaspirate material). It is possible to measurein real time the number of PASCs injected into the patient, the flow ofthe injected cells, oxygen pressure, angiogenesis levels and otherparameters according to the treatment. One of the advantages of thistechnology is the ability to treat patients suffering acute injury,stroke, massive heart attack, or burn immediately after the injury hasoccurred. There is a critical time frame for tissue repair of thesetypes of injuries. PASCs are therefore ideal cells to treat such acutedisorders. Furthermore, all procedures can be performed in the sameoperating room.

FIG. 7 indicates the capacity of PASCs for self-renewal. Expansion ofPASCs in suspension only requires a gentle pipetting to disaggregatePASC clusters to single cells. These individual cells start formingclusters of cells. Once cell clusters are larger than 50 μm, the processof expansion is repeated again (third generation). PASCs have a lowgrowth rate of 1-½ days/cell division.

FIG. 8 shows that PASCs isolated from lipoaspirate of normal woman shownormal karyotype and 23 pairs of chromosomes including the sexchromosomes XX (female donor).

FIGS. 9A-9B show flow cytometry analysis of surface protein expressionfor clusters of differentation (CD) markers in PASCs. PASCs werecharacterized by using the following CD markers: CD105 (marker ofmesenchymal stem cells), CD29 (marker for T cells), CD90 (marker forthymocytes), CD73 (marker for lymphocyte differentiation), CD34 (markerfor hematopoietic stem cells), CD45 (marker for hematopoietic stemcells), CD44 (marker for activated T lymphocytes), HLA-DR (marker forHLA class I) and HLA-DR (marker for HLA class II). (9A) Graphic barrepresents the percentage of expression of surface markers on MuseCells. (9B) Representative histograms of some clusters of differentation(CD).

FIG. 10A indicates that PASCs form aggregates, along with individualcells, both expressing characteristic pluripotent stem cell markers(SSEA3, Oct3/4, Nanog, Sox2 and TR1-60), ADSCs were used as negativecontrols.

FIG. 10B indicates that all cells surviving the severe cellular stresstreatment are indeed PASCs (see recognition of all cells by thepluripotent stem cell markers SSEA4, Oct3/4, Sox2 and TRA1-60 (seeoverlap between pluripotent stem cell markers, DAPI and total cellsdetected by light microscopy, BW).

FIGS. 11A-11C show PASC differentiation into mesodermal cell lineages.(11A) Spontaneous differentiation of PASCs into a mesodermal lineage wasdetermined by using antibodies to DLK (marker for preadipocytes,BODIPY-C₁₆ (fluorescent dye to detect lipid accumulation), and myosin DHeavy Chain) (marker for the heavy chain portion of the Myosin IIprotein found in skeletal muscle cells). (11B) PASC differentiation toadipocytes in adipogenic medium (3 and 6 days). Lipid accumulation ofnewly formed adipocytes was detected using BODIPY-C₁₆. (11C) PASCdifferentiation to myocytes in myogenic medium (3 and 6 days). Smoothmuscle cells were detected using SMA antibody. ADSCs were used ascontrols.

FIGS. 12A-12B show PASCs differentiation to endodermal cell lineages.(12A) Spontaneous differentiation of PASCs into a mesodermal lineage wasdetermined using α-fetoprotein (a marker for development of endoderm andprogenitors of hepatocytes) and pan keratin (marker for filamentscharacteristic of biliary tract epithelial cell) antibodies (12B) PASCsdifferentiation to hepatocyte like cells was determined in inhepatogenic differentiation medium (3 and 6 days) using and cytokeratin7 (a marker for an intermediate filament protein in biliary cells)α-fetoprotein antibodies. ADSCs were used as negative controls.

FIGS. 13A-13C illustrate PASC differentiation to ectodermal celllineages. (13A) Spontaneous differentiation of PASCs into ectodermalcell lineages was determined using antibodies to neuron-specific enolase(NSE, a marker used to detect neocortical neuron progenitors),metabrotopic-glutamate receptor (Glut-R, a marker used to detectmicroglial and neural like cells) and NeuroD (a marker used to detectneocortical precursor cells). (13B) Morphological progression of PASCsinto neurons throughout the incubations in both the first and secondneurogenic differential mediums. PASCs exhibited a progression from theformation of large cell spheres with finger-like projections to long,neuron-like cells, which subsequently formed large networks. (13C)Neural cells derived from PASC were generated after incubation of PASCsin both first and second neurogenic differential medium. Neural likecells were characterized using nestin (marker of neural progenitor cell)and MAP2 (marker involved in the polymerization of microtubules). ADSCswere used as controls.

FIGS. 14A-14G show that PASCs do not produce teratomas. NODscid micewere intratesticular injected with 10⁶ PASCs for teratoma formationassay. (14A) The cell line F19 was used as positive control formedteratoma after 21 days-post injection. Muse cells did not form teratomaat (14B) 1 month, (14C) 2 months (14D) 4 months, (14E) 10⁵ PASCsinjected into the testis of NODscid mice after 6 months post-injectiondid not form teratomas. (14F-14G) Histological studies of testisobtained from NODscid mice after 6 months post-injection (10⁵ PASCs)shows normal testicular tissue. Arrows indicate PASCs-injectedtesticles. On the left, their respective controls.

FIG. 15 illustrates microarray analysis of functional group of genesthat are expressed in PASCs and not in ADSCs, and vice versa. PASCs showvery low expression of genes associated with mitosis, cell cycle, cellproliferation, cell adhesion, DNA repair, cell survival, ubiquitination,actin remodeling, metabolism and genes. In contrast, PASCs exhibit avery high level of expression of genes related to immunity,inflammation, immune regulation, immune response, immune suppression,lymphocyte activation, T cell marker, T cell proliferation, T cellactivation, co-stimulation. PASCs may regulate dendritic cells orT-helper cells at the site of an infection.

FIG. 16 indicates the top ten functional groups pathways of alldifferentially expressed genes (2 fold or higher) in PASCs versus ADSCs.Fischer's exact test was used to calculate a p-value determining theprobability of the association between the genes in the data set withfunctional groups. Functional groups are displayed along the x-axis,while the y-axis displays logarithm of p values calculated by Fisherexact between the ratio of the number of genes differentially expressedgenes (2 fold or higher) in PASCs vs ADSCs in a given functional groupdivided by total number of genes that make up that functional group orpathway with a threshold for statistical significance set at 0.05. Theanalysis was performed by Ingenuity Pathways analysis software.

FIG. 17 indicates the top ten canonical pathways of all differentiallyexpressed genes (2 fold or higher) in PASCs vs ADSCs. Fischer's exacttest was used to calculate a p-value determining the probability of theassociation between the genes in the data set with canonical pathways.Canonical pathways are displayed along the x-axis, while the y-axisdisplays logarithm of p values calculated by Fisher exact between theratio of the number of genes differentially expressed genes (2 fold orhigher) in PASCs vs ADSCs in a given canonical pathway divided by totalnumber of genes that make up that canonical pathway with a threshold forstatistical significance set at 0.05. The analysis was performed byIngenuity Pathways analysis software.

FIG. 18 shows that MicroRNA Let-7 is the most significant upstreamregulator present in PASCs versus ADSCs. Let-7 regulates 11 downstreamgenes associated with decrease of cell cycle division (e.g. CDCA3,CDC16), cell differentiation (DZIP1), cellular growth and proliferation(SSR1), DNA replication (MCM6), replication factor and cancer (RFC3,RFC5) and cell death and survival (NUF2, BRCA1, BUB1B, CDC16.Over-expression of Let-7 in PASCs would potentially play a critical rolein inhibiting Lin28 expression, and therefore would protect these cellsfrom tumorigenic proliferation and teratoma formation.

FIGS. 19A-19C show that PASCs have phagocytic activity. Formation ofPASCs were detected after co-culture of adipocyte fraction (floatingcells) and the stromal vascular fraction (adherent cells). There is aclear interaction between preadipocytes (S-100 (+) cells) and PASCsindicating by (CD34(+) cells, marker of hematopoietic stem cells) whichcould be the smallest and most primitive PASCs that appear to be formed.(19A) PASCs are firmly attach to approximately a third of thepreadipocytes. This attachment is apparently selective since seeminglyidentical preadipocyte cells are completely engulfed in PASCs. (19B)Preadipocyte undergoing mitosis left undisturbed by PASCs while thepreadipocyte directly above is engulfed by PASCs leaving only the DAPIpositive nucleus uncovered. While both preadipocytes and PASCs areS-100+ and SSEA3, (FIG. 19C-a, 19C-b) indicate that peadipocytes wouldshow positive S-100 staining with fluorescent light exposure of 1-2seconds, with PASCs would display positive results with only a 1millisecond exposure time. The great discrepancy in exposure strengthssuggests that the PASCs express S-100 at much higher levels, or morereadily allows S-100 anti-body bind to its domain. Individual PASCs wereoriginally identified through the clear DAPI staining of their smallnuclei. The preadipocytes could clearly be seen at different stages ofbeing phagocytized. Unlike macrophages that phagocytize entire cells,PASCs leave the nucleus of preadipocytes untouched, (FIG. 19A) oftentimes resulting in preadipocyte nuclei that are left bare in culture(FIG. 19B).

FIGS. 20A-20E illustrate the role of PASCs as antigen-specificimmunomodulatory cells. Splenocytes of NOD BDC2.5 transgenic micecontain CD4 (+) T cells, which only recognize a specific peptide namedMimetope (Mim) whose sequence is part of chromogranin A (ChgA).Splenocytes (5×10⁵ cells) were cultured in 24-well clusters withMimetope for 72 hours in the presence (co-culture) or absence of PASCs(10³-10⁵ cells/well). The number of splenocytes (cell proliferation) wasdetermined by FACS on stained carboxyfluorescein-diacetate succinimidylester (CFSE) splenocytes before and after incubation with Mim±PASCs.IFNγ production was determined by FACS and RIA respectively. Co-cultureof PASCs with NOD BDC2.5 transgenic mice splenocytes stimulated with Mimsignificantly reduced the secretion of INFγ by Ag-specific stimulatedsplenocytes. The histograms show dilution of CFSE in T CD3⁺ gated cells.T cell proliferation was not affected by PASC conditioned media. (20A)NOD BDC2.5 splenocytes (spl) were stimulated with a mimotope (Mim) andculture for 72 h without and with (20B) 10⁵ PASCs, (20C) 10⁴ PASCs and(20D) 10³ PASCs. (20E) INFγ secretion by splenocytes stimulated with Mimwas dramatically diminished by the presence of PASCs in the culture.

FIGS. 21A-21D illustrate that PASC conditioned medium contains factors(cytokines, growth factors, peptides) that play a critical role inantigen-specific immunomodulation. NOD BDC2.5 transgenic micesplenocytes were stimulated with a Mimetope (Mim) and cultured withseveral indicated dilutions of PASC conditioned media for 72 hours. PASCconditioned media significantly reduced the secretion of INFγ byAg-specific stimulated splenocytes. An ELISA kit was used to quantifymouse INFγ. Conditioned medium of PASCs isolated from two differentpatients (PASC-CM #1 and PASC-CM #2) t were kept in culture for 72hours. NOD BDC2.5 splenocytes were stained withcarboxyfluorescein-diacetate succinimidyl ester (CFSE), incubated withMim for 72 h and CFSE dilution was analyzed by FACS as measured of cellproliferation. The histograms show dilution of CFSE in T CD3⁺ gatedcells. T cell proliferation was not affected by PASC conditioned media.(21A, 21C) effects of PASC-CM #1; (21B, 21D) effects of PASC-CM #2.

FIGS. 22A-22C show the effect of PASCs in a diabetic mouse model. (22A)NODscid mice were made diabetics by multiple (45 mg/kg, four consecutivedays) injections of streptozotocin (STZ). PASCs were injected (10⁶cells, i.p.) into diabetic (glycemia >240 mg/dl) NODscid mice once ortwice (as indicated). Glycemia and body weight were registered once aweek. (22A) Most of PASCs-treated mice survived for a prolonged periodmaintaining an increase in body weight gain until nearly 6 weeks. Sixout of seven mice survived 8 weeks-post treatment and five out of seven,reached week-11 post-treatment. (22B) Most of the PASCs-treated micemaintained oscillating glycemia below 500 mg/dl at least during 6-weekspost-treatment. (22C) All diabetic NODscid mice receiving PBS (controlgroup) had glycemia ≥500 mg/dl by day 5 and, died or were moribund atday 6 after first injection.

FIGS. 23A-23C show the effect of PASCs in a naturally occurring diabeticNOD mouse model. Soon after the onset of hyperglycemia, PASCs wereinjected (10⁶ cells, i.p.) into NOD mice. (23A) Three out of fourPASCs-injected NOD diabetic mice showed an increase in body weight gainuntil 5-weeks post-treatment. (23B) Blood glucose showed oscillatinglevels with a marked delay to reach ≥500 mg/dl during a prolongedperiod. (23C) All diabetic NOD mice receiving PBS (control group)reached blood glucose levels ≥500 mg/dl (day 4) with constant loss ofbody weight. At day 10, NOD diabetic mice died (n=2). These resultsindicate that PASCs-treated mice survived for prolonged periods andexhibited a significantly higher survival rate than controls.

FIG. 24 represents many of the properties of PASCs that make them idealpluripotent stem cells for tissue regeneration and cell therapy. PASCsare intrinsically present in a quiescent state under normalphysiological circumstances within the cellular niche. Multiple adultstem cell lineages have been shown to exist in a quiescent state atvarious time points throughout their lifespan, including hematopoieticstem cells and epithelial stem cells, which quiescence is thought toplay a role in the preservation of their self-renewal. Severe cellularstress (starvation, low temperatures, prolonged incubation with theproteolytic enzyme collagenase) activates PASCs leading to an increasein glycolitic metabolism and high levels of ROS scavengers. Furthermore,PASCs maintains their pluripotency, without undergoing cellproliferation and differentiation. PASCs do not produce teratomas invivo due to in part their low level of expression of oncogenes coupledwith high expression of tumor suppressor Finally, due to their highresistance to cellular stress, PASCs have a high degree of survival andregenerate damaged tissue with very high efficiency.

FIG. 25 illustrates the potential of PASCs to become cancer stem cells(CSCs) if they are activated under abnormal stress conditions (e.g.ionizing radiation, ultraviolet light, chemical compounds, error-proneDNA repair, etc). Similarly, programming PASCs with the Yamanaka'sfactors lead with the formation of iPS cells (cells with teratogenicpotential). It may be possible to activate endogenous PASCs, which couldaccount for the small population of cells that are converted into iPScells. Such a theory is supported by previous studies regarding thepossible role of adult organ-specific positive Oct4 (+) stem cellsduring asymmetric division in the generation of cancer cells. CSCs maypotentially be generated by PASCs under abnormal stimulus. They maydivide very quickly, with a very active glycolytic metabolism. CSCs canstimulate premature differentiation, cellular death, increase oncogeneexpression and exhibit low expression of tumor suppressor driving tumorgrowth. Furthermore, potential CSCs derived PASCs are resistant tochemo- and radiotherapy and are responsible for its relapse aftertreatment.

DETAILED DESCRIPTION

The invention described herein overcomes major clinical limitations,particularly addressing the problems posed by autologous transplantationwith pluripotent stem cells that do undergo teratogenesis in vivo, andthe need for a high post-transplantation survival rate. Hostile hostfactors of the engraftment microenvironment such as hypoxia, nutritiondeprivation, pro-inflammatory cytokines, and reactive oxygen species caneach contribute to unwanted differentiation or apoptosis.

The invention provides the isolation and characterization of a newpopulation of adipose tissue (AT) derived pluripotent stem cells, termedPluripotent Adipose Stem Cells (“PASCs”). PASCs are isolated usingsevere cellular stress conditions, including long-term exposure to theproteolytic enzyme collagenase, serum deprivation, low temperatures andhypoxia. Under these conditions, a highly purified population of PASCsis isolated without the utilization of cell sorting methods, magneticbeads or special devices and prolonged cell culture procedures.

The complete process of liposuction, collagen digestion of the aspiratedfat, followed by PASCs isolation (100,000,000-200,000,000 PASCs/l oflipoaspirate material), and injection of PASCs (via i.v. or into thedamage tissue) takes about 5-7 hours. This procedure is extremely fastand efficient, making it particularly useful in cases of acute injury,stroke, massive heart attack, or burn, in which it is critical to beginthe treatment of damaged tissue by enhancing the tissue environment forrapid healing and restoration of function. All procedures can beperformed in the same operating room.

Harvesting human adipose tissue by lipoaspiration is a safe andnon-invasive procedure (24), and hundreds of millions of adipose cellscan be isolated from 1-2 liters of lipoaspirate material (25). Becausethis procedure takes less than an hour, adipose tissue could prove theideal source for Muse cell isolation as opposed to bone marrow ordermis.

Using lipoaspirate material, the invention provides a novel methodologyfor the isolation of a population of human Muse cells under severecellular stress conditions (e.g., long term incubation with proteolyticenzyme, 4° C., serum deprivation, and/or hypoxia). Purification of humanPASCs does not require the use of cell sorting, magnetic beads, specialdevices, or cell culture procedures.

PASCs can grow either in suspension, forming cell spheres, similar tohuman ES cell-derived embryoid bodies. However, in adherent culturedishes, PASCs initially form aggregates that start to differentiate intoprogenitors and mature cells, losing their pluripotency as previouslyreported (26). Immunocytochemistry studies demonstrate that PASCsexpress pluripotent stem markers including SSEA3, TR-1-60, Oct3/4, Nanogand Sox2 (26).

PASCs can spontaneously differentiate into mesenchymal, endodermal andectodermal cell lineages with an efficiency of 23%, 20% and 22%,respectively (26). Interestingly, PASCs preferentially differentiatespontaneously into adipocytes (61%), suggesting that PASCs have anepigenetic memory of their tissue of origin. It may be possible thatadipose tissue acts as a natural reservoir for the cells, and withoutstress PASCs may remain in a dormant state (26-28).

Upon introduction to specific culture conditions, PASCs candifferentiate to mesenchymal (adipocytes, skeletal and smooth musclecells), endodermal (hepatocytes and biliary ducts) and ectodermal(neural cells) cell lineages with an efficiency between 80-90%.

Microarray data confirmed that PASCs over-express the pluripotent stemcell markers SOX2, OCT3/4, (POU5F1) and REX1 3-4 fold in comparison topreviously studied multipotent adipose stem cells (ASCs), indicating theintrinsic pluripotent and differential capacity of PASCs. Concordantly,PASCs exhibit up-regulation of genes associated with embryonicdevelopment, albeit at a much lower level than is observed for hESCs oriPSCs.

Microarray analysis reveals that PASCs highly express genes involved incellular protection against oxidative stress. For example, PASCsover-express ALDH1A2 (47-fold change versus ASCs) and SOD2 (41-foldchange versus ASCs) which have anti-oxidative stress and anti-apoptoticfunctions (27, 28). Additionally, these cells also exhibit up regulationof CXCL2 gene expression, a critical chemokine involved in stem cellhoming (29-30).

PASCs have a relatively low expression of many genes involved in tissuedevelopment, cellular assembly and organization, cellular function andmaintenance, DNA replication, repair, and cell cycling in comparisonwith ADSCs. These results suggest an intrinsic non-tumorigenic capacityof PASCs, similar to previously published data of the regenerativeproperties of Muse cells in the absence of the production of teratomasupon transplantation in vivo (22-23). However, under abnormal stressconditions (e.g. programming Muse cells with the Yamanaka's factors), itmay be possible to activate endogenous Muse cells, which could accountfor the small population of cells that are converted into iPS cells(23). Such a theory is supported by previous studies regarding thepossible role of adult organ-specific positive Oct4 (+) stem cellsduring asymmetric division in the generation of cancer cells (31).

Many genes related to DNA repair are up-regulated in PASCs, indicating apotentially high capacity to resist DNA damage as a result of severecellular stress. Furthermore, several ABC-cassette genes weredifferentially expressed in PASCs, indicating that active expression ofdrug transporter genes may play a role in observed stress resistance.

Many of the differentially expressed genes in PASCs are highlyconserved, with homologues present in numerous small organisms (yeast,S. Cerevisiae, C. elegans, chlamydomonas, T. californica, drosophila,etc.). This indicates the possibility that PASCs play a role in a highlyconserved cellular mechanism related to cell survival in response tosevere cellular stress (32-33).

In various embodiments, new methods are provided for the isolation andproliferation of a population of pluripotent stem cells. These cells canbe isolated with a high degree of purity (>90%) by using a simple yethighly efficient purification technique involving severe cellular stressconditions. The methods described herein provide a significant number ofcells without the need of cell sorting, nanobeads techniques andlong-time cell expansion. Moreover, lipoaspiration is a safe andnon-invasive procedure that allows for rapid procurement of suitablesource material for therapeutic applications.

Without being bound by a particular theory, it is believed that thecells that give rise to pluripotent adipose stem cells (PASCs) isolatedaccording to the methods described herein, are small cells that arepresent in adipose tissue in both adipocyte fraction (top layer, PASCsare surrounding adipocytes) as well as in the stromal vascular fraction(SVF). The adipose SVF contains different cell types including adiposetissue macrophages (ATMs), multipotent adipose stem cells (ADSCs),endothelial cells, pericytes among others.

PASCs, prior to cellular stress disruption, are present in a quiescentstate under normal physiological circumstances within the cellular niche(34-35). Multiple adult stem cell lineages have been shown to exist in aquiescent state at various time points throughout their lifespan,including hematopoietic stem cells and epithelial stem cells, whichallegedly plays a role in the preservation of their self-renewal(36-37).

Paracrine/autocrine interactions between the adipocyte fraction and thecellular components present in the SVF could be critical contributors todisruption of the quiescent stage (normal physiological circumstances),and inducing the release and activation of PASCs from both fractions.This paracrine interaction involves cell-cell contact as well assecretion of soluble factors (cytokines, growth factors, etc), whichcould play a significant role in such an activation process.

Definitions

The term stem cells (stammzellen), established over a century ago byGerman scientist Ernst Haeckel (38-40), refers to cells with thecapacity of self-renewal and the ability to differentiate into variouscell types. Based on their ability to give rise to adult tissues of thethree embryonic germ cell lineages, there are different types of stemcells.

The term ‘unipotent’ stem cells” refers to cells having the ability todifferentiate into one cell type, for example, a muscle stem celldifferentiating into a mature muscle cell.

The term “oligopotent stem cells” refers to the capacity of such cellsto differentiate into few, but not all, cell types within a specifictissue (e.g. lymphoid or myeloid stem cells) (41).

The term “multipotent stem cells” refers to the ability of such cells todifferentiate into all cell types from a specific germ layer, whichincludes mesenchymal stem cell (MSC) (42-43). For example, adiposederived stem cells (ADSCs) are multipotent stem cells that have thepotential to differentiate only into mesenchymal cell lineages,including adipocytes, chondrocytes, osteoblasts, and myoblasts in vitroand to undergo differentiation in vivo, in a manner similar to that ofmultipotent stromal cells derived from bone marrow (24, 44).

The ‘totipotent’ stem cell is the most primitive stem cell, mostcommonly known as the zygote, capable of differentiation into embryonicand extraembryonic cell types (placenta) and even create an entireorganism (45-46).

By “pluripotency” and “pluripotent stem cells” or “PSCs”, it is meantthat such cells have the ability to differentiate into all types ofcells in an organism. Pluripotent cells are characterized by theexpression of several pluripotency markers known by one of ordinaryskill in the art. Such markers include, but are not limited to AlkalinePhosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1,Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. In certain embodimentspluripotent cells are capable of forming or contributing to ectoderm,mesoderm, or endoderm tissues in a living organism.

Because embryonic stem cells (“ESCs”) and induced pluripotent stem cells(“iPSCs”) exhibit self-renewal that is frequently uncontrolled and oftenmaterializes in teratoma formation, cell pluripotency is oftenerroneously defined as the inherent capacity of cells to differentiateinto all types of cell and to produce teratomas.

As used herein, “PASCs (pluripotent adipose stem cells) are positive forSSEA-3, SSEA-4, Oct 3/4, SOX-2, TRA1-60, and NANOG. PASCs are isolatedfrom adipose tissue under severe stress conditions, and aredistinguished from iPSCs and ESCs in that the PASCs described hereinexhibit both low proliferative and telomerase activities, a normalkaryotype, as well as asymmetric growth, and do not undergotumorigenesis or teratoma formation when transplanted into a hostorganism (e.g. immunodeficient mice). In addition, hES and iPS cellsexpress the above-listed markers at a level that is many thousand-foldhigher than the low level of expression observed in PASCs. PASCs differfrom Muse (multilineage differentiating stress-enduring) cells in theirexpression of cell markers. PASCS have the capability to differentiateto any type of cell of mesodermal, endodermal or ectodermal origin.

The term “multipotent stem cells” refers to the ability of such cells todifferentiate into all cell types from a specific germ layer, whichincludes the increasingly popular mesenchymal stem cell (MSC) (42-43).For example, adipose-derived stem cells (ADSCs) are multipotent stemcells that have the potential to differentiate only into mesenchymalcell lineages including adipocytes, chondrocytes, osteoblasts, andmyoblasts in vitro and to undergo differentiation in vivo, in a mannersimilar to that of multipotent stromal cells derived from bone marrow(24, 44).

Muse (Multi-lineage differentiating stress enduring) cells arepluripotent stem cells present in mesenchymal tissue of the body. Thesecells are highly resistant to cellular stress. Muse cells areSSEA3(+)/CD105(+) cells isolated by cell sorting and without use ofstress conditions. Muse cells are able to generate cells representativeof all three germ layers and do not undergo teratoma formation whentransplanted into a host environment in vivo. (22-23).

The terms “primary cells”, “primary cell lines”, and “primary cultures”are used interchangeably herein to refer to cells and cell cultures thathave been derived from a subject and allowed to grow in vitro for alimited number of passages, i.e. splittings, of the culture. Forexample, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary adiposecells of the present invention are maintained for fewer than 10 passagesin vitro prior to use.

As used herein, “co-culturing” of cells refers to maintaining two ormore populations of cells in contact with each other such that theconditions permit autocrine and/or paracrine interactions between thecell populations. The co-culturing environment can be a conventionalcell culture environment, or it can be incubation or storage of the cellpopulations in a common vessel, such as a centrifuge tube or cultureflask.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human or veterinary subject, and includes: (a)preventing the disease or symptom from occurring in a subject which maybe predisposed to the disease or symptom but has not yet been diagnosedas having it; (b) inhibiting the disease symptom, i.e., arresting itsdevelopment; or (c) relieving the disease symptom, i.e., causingregression of the disease or symptom.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans. Alsocontemplated are veterinary subjects, including, but not limited to,horses, cattle, dogs, cats, sheep, pigs, goats, rabbits, and rodents.

By “adipose” is meant any fat tissue. The adipose tissue may be brown orwhite adipose tissue, derived from, omental/visceral, mammary, gonadal,or other adipose tissue site. In certain embodiments the adipose issubcutaneous white adipose tissue or visceral adipose tissue or anyother tissue containing adipose cells. The adipose tissue may be fromany organism having fat tissue. Preferably, the adipose tissue ismammalian, most preferably the adipose tissue is human. A convenientsource of adipose tissue is from liposuction surgery, however, thesource of adipose tissue need not be so limited.

For isolation of primary cells from tissue, an appropriate solutioncontaining collagenase (0.1%) may be used for dispersion or suspension.Such solution will generally be a balanced salt solution, e.g. Iscove'smodified DMEM, normal saline, PBS, Hank's balanced salt solution, etc.,in conjunction with an acceptable buffer at low concentration, generallyfrom 5-25 mM. Convenient buffers include HEPES, phosphate buffers,lactate buffers, etc. Collagenase digestion releases all cells presentin adipose tissue, including (i) the adipocyte fraction (floating cellsin the top layer) and (ii) the stromal vascular fraction (cells able toprecipitate after centrifugation, e.g. adipose tissue macrophages,adipose derived stem cells, endothelial cells, fibroblasts). PASCs arepresent in both the adipocyte and stromal vascular fractions. Thisdigestion is typically performed by incubation of the lipoaspiratematerial with an equal volume of DMEM containing 0.1% collagenase for30-45 min at 37° C. in a shaking incubator at 110 rpm.

“Severe cellular stress” includes, but it is not limited to, exposingcells to collagenase, a proteolytic enzyme that breaks the peptide bondsin collagen. Collagen is a vital part of the extracellular matrix inflesh and connective tissue in the animal body. In contrast to serineproteases (trypsin, chymotrypsin, and elastase), collagenase does notundergo self-degradation, making a very effective protease. Severecellular stress also includes exposing cells to starvation conditions(no nutrients, no glucose), hypoxic conditions (lack of oxygen), lowtemperatures, heat shock, and lysis by mechanical procedures such assonication.

Methods of Isolation of PASCs

One non-limiting approach to the differentiation and proliferation ofPASCs can be performed as described below and schematically illustratedin FIGS. 4-5. Briefly, adipose tissue can be collected by lipoaspirationfrom a subject (e.g., a human, a non-human mammal) of any sex, age andrace under local anesthesia. The tissue is harvested from subcutaneousabdominal tissues or/and other part of the body containing fat (e.g.tights) by simple procedures, e.g., manually by using specific syringes,or using an automatic lipoaspirator following standard protocols. Bloodthat normally is presented in the lipoaspirate material is removed byseveral washes with PBS (or other buffer). The lipoaspirate material isthen subjected to centrifugation to remove oil (top lawyer) andtumenescent solution (low lawyer) from the adipose tissue (middlelawyer). The concentrated adipose tissue is then digested with aproteolytic enzyme, such as 0.02-1% collagenase, typically 0.1%collagenase, and incubated at 37° C. for 30 minutes using a shakerincubator at 100 rpm. Digested material is then kept at 2-12° C.,typically at 4° C., for about 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26,28, or 30 hours, preferably for about 6-8 hours (FIG. 5). During thatprocess, samples can be slightly shaken (20-30 rpm) or rotated in angle(90°-180°, to maximize cell-cell contact between adipocyte fraction andSVF fraction). Hypoxic conditions typically means an environment of0.01% to 10% oxygen. In one embodiment, the hypoxic condition is anenvironment of 0.01% to 5% oxygen, such as, for example, about 1%oxygen.

In some embodiments, the proteolytic enzyme is a serine protease enzymes(e.g., trypsin, chymotrypsin); aspartate protease (e.g. pepsin); cysteinprotease (e.g. papain, chymopapain); metalloproteinases (e.g.thermolysin, glutamate protease). Concentrations of these proteolyticenzymes vary according to their efficiency to activate and releasePASCs. Severe cellular stress conditions could also include vortexingthe cells for short (30 seconds) to long periods of time (10 min),optionally including additional proteolysis. Other alternative methodsfor severe cellular stress conditions include sonication of the cells(externally) at different power potential (1-50 kHz) at different times(1 min to 1 hour) and/or different temperatures (4-37° C.), optionallyincluding additional proteolysis.

The resuspended material is then centrifugated at 1,000 rpm/4° C./10min. The adipocyte fraction (floating cells) is removed by aspirationfrom the stromal vascular fraction (cell pellet). This stromal vascularfraction contains living PASCs that are resistant to the longcollagenase/lack of nutrients/4° C./hypoxia treatment (FIG. 5) and otherdeath cell components, non-resistant to such environment includingADSCs, adipose tissue macrophages, endothelial cells (FIG. 5). Thesedead cells are removed from the PASCs by successive washes, e.g., inDMEM/5% FCS/antibiotics. Highly purified PASCs can grow as individualcells or by forming cell clusters in suspension, or attached to a cellculture dish (FIG. 5). PASCs maintain their pluripotent characteristicsindefinitely. Isolation, differentiation and proliferation of PASCs canbe easily performed by any cell biologist (Ph.D., Master in Science,technician) after receiving the appropriate training.

In various embodiments, methods are provided for the isolation andproliferation of a population of pluripotent adipose stem cells (PASCs).In contrast to induced pluripotent stem cells, the pluripotent adiposestem cells of the invention can be isolated without the use of geneticmanipulation. Moreover, the methods described herein provide asignificant number of cells without the need for long time cellexpansion.

In certain embodiments, human adipose tissue is used as a source ofpluripotent adipose stem cells because cell isolation from this tissuerequires a minimally-invasive technique. In adipose tissue, differentcell types (adipose derived stem cells, progenitors, and mature cells)are highly abundant. However, the methods described herein need not belimited to the use of adipose tissue and other tissues (e.g., bonemarrow, skin, blood, etc.) can similarly be used. Harvesting cells fromadipose tissue offers significant advantages over cell isolation frombone marrow. The harvesting procedure of cells from adipose tissue isless painful and many more cells may be harvested. For example, thenumber of ADSCs (mesenchymal origin) present in adipose tissue is 100 to1000 times higher per milliliter than that of mesenchymal stem cells inbone marrow (26).

In one embodiment, the invention provides a method of isolatingpluripotent adipose stem cells (PASCs) from adipose tissue. The methodtypically comprises the steps of: (a) providing an adipocyte tissuesample; (b) subjecting cells in said sample to initial stressconditions; (c) co-culturing adipocytes and a stromal vascular fractionfor 2-36 hours; (d) recovering the viable cells; and (e) optionallyculturing the recovered cells. In one embodiment, the stress conditionsof step (b) comprise incubating the cells in a medium containing aproteolytic enzyme. The co-culturing of step (c) can be performed in thepresence of a proteolytic enzyme as well. In a more specific embodiment,the enzyme is collagenase.

The adipose tissue sample may be obtained via means known to thoseskilled in the art, including lipoaspiration. In one embodiment, thelipoaspiration is performed on a subject to whom the isolated PASCs willbe administered. Alternatively, the lipoaspirate may be obtained from asuitable donor, preferably one allogeneic with the recipient. Typically,the sample is used immediately upon collection from the donor, usuallywithin about 1-2 hours of collection. Optionally, the lipoaspiratematerial can be kept at 4° C. up to 48 hours after the lipoaspirationprocedure. Considerations of histocompatibility are of greatest concernwhen the PASCs are to be administered to a host for therapeuticpurposes. Such considerations are of lesser or no importance to otherapplications, such as some cases of experimental use, screening ortesting, and where conditioned medium (and not cells) will beadministered to the recipient. Preliminary data, however, show thatallotransplantation (transplant of PASCs from one individual to another)can be performed without immunorejection by the host individual.

PASCs isolated from adipose tissue may be cultured in vitro undervarious culture conditions. PASCs are typically grown asundifferentiated pluripotent stem cells in non-adherent culturedishes/flasks. Culture medium may be liquid or semi-solid, e.g.containing agar, methylcellulose, etc. The cell population may beconveniently suspended in an appropriate nutrient medium, such asIscove's modified DMEM or RPMI-1640, normally supplemented with fetalcalf serum (about 5-10%), L-glutamine, a thiol, particularly2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.Because PASCs have a mesenchymal origin, various custom cell culturemedia designed to grow mesenchymal stem cells under optimal conditionscan be utilized. These special culture media are commercially availablefrom companies such as BD, Stem Cell Technology, and the like.

In one embodiment, the adipose cells are maintained in culture in theabsence of feeder layer cells. In certain embodiments the culture maycontain growth factors to which the cells are responsive. Growthfactors, as defined herein, are molecules capable of promoting survival,growth and/or differentiation of cells, either in culture or in theintact tissue. Growth factors include polypeptides and non-polypeptidefactors.

PASCs can be more easily isolated than Muse cells (another pluripotentstem cell isolated under stress conditions) because isolation of PASCsdoes not require cell sorting, culture procedures.

The stress conditions of step (b) include, but are not limited to,protease treatment, exposing cells to starvation conditions (nonutrients, no glucose), hypoxic conditions (lack of oxygen), lowtemperatures, heat shock, and lysis by mechanical procedures such assonication. Two or more such stressors may be used together. The stressconditions are typically applied for 20-60 minutes, and may continuethrough the co-culturing step. For example, the cells obtained fromadipose tissue can be treated initially with protease (e.g.,collagenase) for about 45 minutes at 37° C. (step (b)), followed bystorage in hypoxic conditions at 4° C. for 2-8 hours (or overnight; step(c)), during which time the proteolytic activity is permitted tocontinue. In another embodiment, the stress conditions compriseincubating the adipose tissue in collagenase for 30-45 minutes at 37°C., followed by 6-8 hours co-culturing of the cells at 4° C.(refrigerator). The decrease of temperature of the cells from 37° C. to4° C. is, in one embodiment, gradual (around 1° C. every 7-8 min).

In a typical embodiment, the co-culturing is performed in the absence ofserum. For example, the co-culturing can be performed in Dulbecco'sMinimum Essential Medium (DMEM), or a similar basic medium known in theart. The co-culturing can be as simple as storing the cells in acentrifuge tube without or with slight shaking or angle rotation.Stressors can be applied, for example, by closing the cap of the tube tocreate hypoxic conditions, placing the tube or other container into arefrigerator (cooling), as well as using a nutrient-free medium(starvation).

In one embodiment, the co-culturing is performed for 4-24 hours. Thelength of the co-culturing can be modified to accommodate timingconsiderations. For example, if isolated PASCs are required on an urgentbasis, such as for treatment of traumatic injury, steps (b) and (c) canbe shortened, and multiple stressors applied, to accelerate the processof selecting for stress-resistant cells.

In one embodiment, the recovering of step (d) comprises recovering atleast 200,000 PASCs. In other embodiments, the recovering comprisesrecovering at least 50,000, 100,000, 500,000 PASCs, or at least1,000,000 PASCs. These quantities of PASCs are typically recoverablewithin 24 hours of initiating the stress conditions of step (b). In someembodiments, at least 500,000 PASCs are recovered within 8 hours ofinitiating the stress conditions of step (b). In other embodiments, thePASCs are recovered within one week. In one embodiment, the method isperformed without cell-sorting. Typically, 100,000,000 PASCs can beobtained from 1 liter of lipoaspirate material 6 hours afterlipoaspiration. PASCs are normally present as individual cells or formclusters, and can grow in suspension as well as form adherent cells(FIG. 5).

Cells, Compositions & Conditioned Media

PASCs are highly abundant in the adipocyte and stromal vascular fractionobtained from adipose tissue (FIG. 1). PASCs have the potential to beused for autologous tissue engineering and regenerative medicine inhumans. Furthermore, PASCs have the potential to not only differentiateto progenitors, but also to mature cells from the three germ line cells(any kind of cell present in the body). PASCs can be used for thetreatment of many disorders as well as for the testing of new drugs.

In one embodiment, the PASCs, such as those recovered using theisolation method described here, are SSEA-3 and/or CD105 positive. Insome embodiments, the cells express one or more markers of pluripotencyselected from the group consisting of SSEA3, Nanog, Oct3/4, Sox2, Klf4,and TR1-60. In some embodiments, the cells express one or more markersof endodermal progenitor cells, such as Cytokeratin 19, HNF-3alpha/FoxA1, EOMES, HNF-3 beta/FoxA2, FABP1/L-FABP, SOX7, FABP2/I-FABP,SOX17, GATA-4, TCF-2/HNF-1 beta, and Goosecoid. In some embodiments, thecells express one or more markers selected from the group consisting ofalpha-Fetoprotein/AFP, HNF-4 alpha/NR2A1, beta-Catenin, MIXL1, GATA-4,SALL4, GATA-6, SOX7, GDF-1, SOX17, and GDF-3. In other embodiments, thecells express one or more markers of mesodermal progenitor cells, suchas EpCAM/CD326, NCAM/CD56, cardiac sarcomic actin, and BODIPY C-16. Inyet other embodiments, the cells express one or more markers ofectodermal progenitor cells, such as BMP-4, Noggin, Chordin, Otx2,FGF-8, p63/TP73L, FoxJ3, Pax2, GBX2, Pax6, Nestin, and beta-III Tubulin.

Adipose tissue is important in metabolic homeostasis through its role asan energy depot and endocrine organ (47-48). Adipose tissue is composedof adipocytes (mature cells), the stromal vascular fraction containingadipose tissue macrophages (ATMs), mesenchymal stem cells named adiposestem cells (ADSCs) and the population disclosed herein, namedpluripotent adipose stem cells (PASCs), which are highly abundant inboth the adipocyte and stromal vascular fractions, among othercomponents (FIG. 1).

Adipogenesis, the process by which ADSCs are converted to adipocytesinvolves three major stages: (i) hASC commitment to the preadipocytephenotype, characterized by the formation of conversion of typicalspindle-shape cells to more rounded cells containing cytoplasmic lipidinclusions (pre-adipocytes); (ii) early-stage preadipocytedifferentiation to adipocytes (immature adipocytes), and (iii)late-stage preadipocyte differentiation into adipocytes, characterizedby cell-cycle arrest with further activation of PPARγ and C/EBPα. Thesepro-adipogenic events are further modulated by other transcriptionalfactors to promote gradual intracellular lipid accumulation andmaturation into adipocytes (FIG. 2) (49-52).

ADSCs are multipotent stem cells that have the potential todifferentiate only into mesenchymal cell lineages including adipocytes,chondrocytes, osteoblasts, and myoblasts in vitro and to undergodifferentiation in vivo, in a manner similar to that of multipotentstromal cells derived from bone marrow (24-25, 44) (FIG. 2).

It is generally accepted that mature adipocytes do not regularly undergomitosis, and thus, an increase in adipocytes usually reflects adifferentiation of preadipocytes (24-25). However, several studiesindicate that mature adipocytes could also have proliferative activity(53-54). Recent studies suggest adipocytes can dedifferentiate topreadipocytes [21] and can even differentiate to a multipotent cellpopulation (FIG. 2) (55-56). Of note, adipocyte precursors andpreadipocytes have also been recently observed to rapidly andefficiently differentiate into typical macrophages (57-58). (FIG. 2).Some reports indicate, although with very low efficiency. Adipose tissuemacrophages (hematopoietic stem cell origin) can differentiate topreadipocytes (mesenchymal stem cell origin) (59). (FIG. 3). Theseresults indicate the high degree of cell plasticity in the differentcell components of adipose tissue.

The invention further provides PASCs as described herein. The PASCs aretypically present in a composition that is essentially free of othercell types (e.g., at least 90% pure, typically 95% pure, and in someembodiments, at least 99% pure). In an alternative embodiment, the PASCsare present in combination with ADSCs, such as at a ratio of 50:50,75:25, 25:75, or other ratio, as desired. ADSCs are currently used forsoft tissue regeneration (face, breast, etc). Because PASCsdifferentiate much faster into fat and muscle cells than ADSCs (at least3-fold faster), they would be more efficient than ADSCs. for suchtreatments. Combination between PASCs and ADSCs would be one option forcombining the features of both cell types. Because PASCs cells arealready adapted to severe cellular stress, similar to the environment ofdamaged tissue, they have a high rate of survival after transplantationand high grafting efficiency, which is critical for tissue regeneration.

Also provided is a composition comprising PASCs isolated according tothe method described herein, or progeny thereof (including a cell linederived therefrom), and a therapeutically acceptable buffer solution,wherein the PASCs are suspended in a physiologically compatible buffersolution (e.g. PBS). Also provided is a composition comprisingconditioned medium that has been recovered from a culture of PASCsisolated in accordance with the methods described herein. Strategies forpreparing therapeutic conditioned media are described, for example, inBhang et al. (60). Culture conditioned media contains various factors(cytokines, chemokines, growth factors, peptides, proteins) secreted byPASCs that have been maintained in culture for a period of about 24-72hours. These factors have anti-inflammatory and/or immunomodulationproperties that could be used for treatment of many diseases, especiallythose related to immunological or autoimmune diseases.

In one embodiment, the composition comprises PASCs isolated fromlipoaspiration. In one embodiment, the lipoaspiration is performed onthe subject to whom the composition is administered. In a typicalembodiment, the composition is administered to the subject within 6hours of the lipoaspiration. In other embodiments, the composition isadministered within 12, 24, or 36 hours of the lipoaspiration.

Methods of Using PASCs

The invention provides a method of ameliorating tissue damage in asubject. The invention additionally provides a method of repairing orregenerating diseased or damaged tissue in a subject. In anotherembodiment, the invention provides a method of enhancing tissue functionand/or a method of delivering cytokines to tissue of a subject.Typically, the method comprises administering a composition of theinvention to the subject. Where the composition comprises PASCs,optionally, it is administered under conditions permitting the PASCs ofthe composition to divide and to populate a site of tissue damage.Migration of PASCs to the site of damage is not required in allembodiments, as factors released by the PASCs provide therapeuticbenefits.

In some embodiments, the tissue damage comprises traumatic injury ordisease-associated damage. Representative examples of traumatic injurycomprise hypoxia, bone injury, laceration, gunshot wound, and spinalcord injury. In another embodiment, the disease-associated damagecomprises damage associated with diabetes, vascular disease, infection,degenerative neurological disease, cancer. In one embodiment, thecomposition is used to regenerate soft tissue, such as for breast andface reconstruction, and other forms of plastic surgery and aestheticmedicine. In another embodiment, the composition is used to regeneratemuscle tissue, such as for treatment of acute injuries and sportsmedicine. In yet a further embodiment, the composition is used to treatimmune and autoimmune diseases, including, for example, Huntington'sDisease, Multiple Sclerosis, Rheumatoid Arthritis, Lupus, Diabetes typeI, Crohn's disease. In another embodiment, the composition is used as ananti-aging reagent, such as by injection in facial and other tissues forrejuvenation. The composition can comprise an extract of proteins,lipids and/or other substances from PASCs for use in cell rejuvenation,prepared as a cream, oil, or other topical application. The compositioncan be a personalized rejuvenation product, prepared from materialisolated from the subject's own body. Based on these potentialapplications of PASCs, the creation of a “Bank of PASCs” could be idealfor use in preventive medicine.

Compositions of the invention can be used to treat injured soldiers.Limb vascular injuries represent 50-70% of all war injuries. Managementof these injuries on the modern battlefield presents many unique anddemanding challenges. Limb vascular injuries are produced by highexplosives, munitions, and high-velocity missiles, often causingsoft-tissue destruction. In these types of surgical interventions, manyservice members experience multiple revision surgeries and are facedwith at least some scarring. Moreover, this type of wounding frequentlyleads to chronic pain and limited motion. Insufficient soft tissuecoverage of bone and other vital structures is a frequent factorcontributing to pain, limited motion and even amputation. Severeischemia produces total necrosis of tissue, but in moderate ischemia,progenitor cells can survive and initiate tissue regeneration. Providingtreatment as soon as possible after injury has not only saved lives, buthas also greatly increased the degree to which limb function can bepreserved for service members wounded in the battlefield. Time toregenerate new tissue and promote neovascularization is critical toavoid cell death and tissue necrosis in the injured area. Providingtreatment as soon as possible after injury has saved lives and preservedoptimal function for most of the personnel wounded in the battlefield.Because of their pluripotency, non-teratogenesis, fast celldifferentiation process, high grafting efficiency, PASCs are ideal cellsto provide injured military personnel with alternatives to prostheticsand to mitigate scarred, nonfunctional healing using patient's ownpluripotent stem cells. PASCs could not only regenerate new tissue, butalso improve the survival of cells within the zone of injury andactively enhance and direct the regenerative capacity of the localtissues to preserve and restore vital body components such as nerve,vasculature and bone, and the fragile soft tissue envelope surroundingthem. The use of PASCs offers the potential to return functional softtissue mass to the craniofacial region and to extremities of injuredsoldiers, and can facilitate their rehabilitation. Furthermore, PASCisolation and transplantation could be readily performed in the sameoperating room located in the battlefield or Level II-treatmentfacility, instead of transporting the injured soldier to a LevelIII-treatment facility.

PASCs have the potential to elucidate new avenues of cancer research,for example, with regards to cancer stem cells, quiescence, malignancyand post-treatment relapse. In some embodiments, the PASCs of theinvention are used in screening for new therapeutic agents, such as foranti-cancer drug screening.

The primary cell population may be used immediately. Alternatively, incertain embodiments, the cell population may be frozen at liquidnitrogen temperatures and stored for long periods of time, being thawedand capable of being reused. In such cases, the cells will usually befrozen in 10% DMSO, 50% serum, 40% buffered medium, or some other suchsolution as is commonly used in the art to preserve cells at suchfreezing temperatures, and thawed in a manner as commonly known in theart for thawing frozen cultured cells.

The isolation of human pluripotent stem cells from adipose tissue opensnew avenues for autologous cell-based therapy that includes tissueengineering, reconstructive medicine and the treatment of degenerativeand immune disorders (e.g. Parkinson's Disease, Alzheimer's Disease,diabetes, Huntington's Disease, etc.) (FIG. 6).

In one embodiment, PASCs are employed as antigen-specificimmunomodulation cells (FIG. 20). Different factors secreted by PASCs(cytokines, growth factors, peptides) could also play a critical role inantigen-specific immunomodulation (FIG. 21).

PASCs significantly improved weight loss and glycemic levels in (i)NODscid diabetic mice and (ii) naturally occurred diabetic miceenhancing the period of survival (FIG. 22-23).

In summary, PASCs can be easily isolated from adipose tissue of anyhuman being (or other mammal) without the use of exogenous genes or cellstress to induce pluripotency. PASCs have the potential to spontaneouslydifferentiate to progenitors and mature cells. Unlike ES and iPSC, it isbelieved that PASCs can regenerate damaged tissues without formingteratomas.

Advantages of PASCs

Muse cells, another pluripotent stem cells resistant to cellular stresshave been isolated be cell sorting, yielding a population of 100% CD105(+)/SSEA3 (+) cells. Muse cells have been hindered by a low yield fromother sources, such as dermal fibroblasts and bone marrow, as Muse cellsmake up only 1-3% of adult tissue. Furthermore, Muse cell isolationrequires not only include cell sorting, but also cell expansionprocedures which requires up to 6 weeks to yield 1,000,000 of Musecells. In contrast, approximately one hundred million PASCs can beextracted from a mere 1-2 liters of tissue, enhancing the number ofextractable PASCs without the need for cell expansion procedures and/orcell sorting.

The isolation procedure, typically a 4-8 hrs digestion in a proteolyticenzyme under serum deprivation, low temperatures and severe hypoxia, isboth time-efficient and cost-effective, negating the necessity for cellsorting techniques, which require expensive reagents and equipment. Thecomplete process of liposuction, collagen digestion of the aspiratedfat, followed by PASC isolation (100,000,000 PASCs/lt of lipoaspiratematerial), and injection of PASCs (via i.v. or into the damage tissue)requires about 7-9 hours.

This procedure is extremely fast and efficient, offering a particularadvantage in cases of acute injury, stroke, massive heart attack, burn,and other urgent conditions in which rapid healing and restoration offunction is critical. All procedures can be performed in the sameoperating room.

Because PASCs are in a dormant stage in both the adipocyte and stromalvascular fractions, interaction of both components (co-culture) isimportant for the activation and release of PASCs. This phenomenoncontributes to the production of very high numbers of PASCs(˜100,000,000 PASCs/ltr of lipoaspirate) in a short period of time (6-8hs). Furthermore, isolation of PASCs form the adipocyte or stromalvascular fraction alone, without co-culture, under the same stressconditions indicated herein, generate a low number of PASCs. Thereforeco-culture of adipocyte and stromal vascular fractions is beneficial forproducing a large number of PASCs.

PASCs express genes associated with cell death and survival, unlikeASCs, indicating a genetic predisposition to the transition from thequiescent to the active state as a consequence of severe cellularstress. The high Let-7/Lin28 ratio present in PASCs protects these cellsfrom teratogenesis despite their pluripotency. The medial to lowexpression by PASCs of many genes involved in tissue development,cellular assembly and organization, cellular function and maintenance,DNA replication, repair, and cell cycling, is indicative of theirintrinsic lack of tumorigenic susceptibility. Moreover, PASCs expressnumerous lymphocytic and hematopoietic genes, such as CCR1 and CXCL2,encoding chemokine receptors and ligands known to be involved in stemcell homing.

PASCs likely function according to a highly conserved cellular mechanismrelated to cell survival in response to severe cellular stress, as wellas the functional regeneration of damaged tissues and amputated limbs inprimitive species. DNA degradation and mutation contribute to theincreasingly harsh milieu of the aging body. As PASCs are inherentlyresistant to cellular stress, and genetically resilient to DNA damage,their application for the investigation of age-related and degenerativediseases is both relevant and promising.

PASCs have phagocytic activity. Unlike macrophages that phagocytizeentire cells, PASCs leave the nucleus of mature cells untouched, whichcould be used by PASCs as genetic information to generate identical andhealthy mature cells. Furthermore, PASCs have the potential to behave asantigen-specific immunomodulation cells by cell to cell contact with Band/T cells. Different factors secreted by PASCs (cytokines, growthfactors, peptides) could also play a critical role in antigen-specificimmunomodulation.

PASCs, are intrinsically present in a quiescent state under normalphysiological circumstances within the cellular niche (34-35). Multipleadult stem cell lineages have been shown to exist in a quiescent stateat various time points throughout their lifespan, includinghematopoietic stem cells and epithelial stem cells, which allegedlyplays a role in the preservation of their self-renewal (34, 35). Severecellular stress (starvation, low temperatures, prolonged incubation withthe proteolytic enzyme collagenase) activates PASCs leading to anincrease in glycolitic metabolism and high levels of ROS scavengers.Furthermore, PASCs maintain their pluripotency, without undergoing cellproliferation and differentiation. PASCs do not produce teratomas invivo, likely due to their low level of oncogene expression tumor andhigh level of tumor suppressor factors expression. Finally, PASCs due totheir high resistance to cellular stress, have a high degree of survivaland regenerate damage tissue with very high efficiency (FIG. 24).

If cellular stress is performed under carcinogenic stress conditions(e.g. ionizing radiation, ultraviolet light, chemical compounds,error-prone DNA repair, etc), PASCs have the potential to become cancerstem cells (CSCs). For example, Muse cells (CD105 (+)/SSEA3 (+)) thatare like PASCs are resistant to cellular stress. Programming of Musecells by the introduction of the four Yamanaka's factors led to theformation of iPS (cells with tumorigenic activity). It may be possibleto abnormal activation of endogenous Muse cells which account for thesmall population of cells that are converted into iPS cells (23). Such atheory is supported by previous studies regarding the possible role ofadult organ-specific positive Oct4 (+) stem cells during asymmetricdivision in the generation of cancer cells (15). Similarly to Musecells, PASCs could have the potential to be converted to CSCs undertumorigenic stimulus. Under these circumstances, PASCs could divide veryfast, with a very active glycolytic metabolism and differentiate intoCSCs. CSC derived from PASCs would have high differentiation, increasein oncogene expression and low expression of tumor suppressor factorsdriving tumor growth. Furthermore, CSCs derived PASCs could be resistantto chemo- and radiotherapy and be responsible for its relapse aftertreatment (FIG. 25). PASCs therefore provide a tool for the developmentof cancer therapies, specifically with regards to identification ofagents for modulation of cancer stem cells, quiescence, malignancy andpost-treatment relapse.

The methods and isolated cells described herein offer the followingfeatures: (i) it is possible to easily isolate pluripotent stem cells(PASCs) from both the adipocyte and stromal vascular fractions undersevere stress conditions; co-culture between both fractions isbeneficial for the production of large amounts of PASCs (100,000,000PASCS/lt of lipoaspirate); (ii) a significant amount of PASCs, asindividual cells and associated in clusters, can be isolated from boththe adipocyte and stromal vascular fractions; (iii) hundreds of clusterscontaining thousands of PASCs can be obtained from a relatively smallamount of adipose tissue; (iv) harvesting adipose tissue is a safe andnon-invasive procedure; (v) from 200-2000 cc of lipoaspirate materialobtained under local anesthesia, it is possible to isolate20,000,000-200,000,000 PASCs; (vi) from the same preparation, it is alsopossible to isolate ADSCs from the stromal vascular fraction (e. g.˜200,000,000 ADSCs/liter of lipoaspirate material); (vii) clusters ofPASCs can be formed within hours, of harvesting the lipoaspiratematerial; (viii) PASCs can be maintained in an undifferentiated state byculturing the cells for many days using non-adherent culture dishes;(ix) progenitors derived from PASCs can be isolated by culturingclusters of PASCs in adherent culture dishes; and (x) mature cells fromthe three germ layers (endodermal, ectodermal and mesodermal) can bealso obtained with low efficiency (20%) by keeping the cluster of cellsin culture for an extended time in adherent culture dishes; mature cellsderived from PASCs can be obtained with very high efficiency (80-90%) bykeeping clusters of PASCs in specific induced differentiation medium.

In summary, PASCs can be easily isolated with a high degree of puritywithout the need of using cell sorting or other methods for cellenrichment that could change the phenotype of PASCs. PASCs candifferentiate to progenitors and mature cells, in a spontaneous processwithout and with the use of an induced differentiation medium. PASCs,unlike ESCs and iPSCs offer the capability to repair and regenerate invivo damaged tissue without forming teratomas.

In addition, it is noted that various advantages of the methodsdescribed herein include, but are not limited to:

(1) Harvesting pluripotent stem cells from adipose tissue bylipoaspiration is less painful than harvesting these cells from bonemarrow cells;

(2) PASCs are spontaneously originated with stress cell treatment andmany clusters of PASCs can be formed from small amount of adiposetissue;

(3) Many PASCs can be highly purified by a simple technique in a veryshort period of time without the need of cell sorting or other cellselection technique;

(4) Because the quick procedure of cell isolation and high yield, PASCscan be reinjected into the patient without the need of cell expansion;

(5) Alternatively, PASCs can be keep in culture and expanded followingstandard cell culture techniques;

(6) It is possible to isolate (i) PASCs, (ii) progenitors derived fromPASCs and (iii) mature cells derived from PASCs which allows celltherapy using either of PASCs/progenitors/mature cells; and

(7) It is possible to screen new drugs using PASCs at different stage ofcell differentiation.

Role of PASCs in Cancer

Tumor growth may be driven by a small population of cells withself-renewal capacity and high tumorigenic potency. These cells arecalled cancer initiating or cancer stem cells (CSCs). These cells aremore resistant to chemo- and radiotherapy, as well as environmentalfactors, They sustain tumor growth and are responsible for its relapseafter treatment. PASCs are highly resistant to cellular stress, andallow for isolation and characterization of such an endogenous cellpopulation that displays cancer like resistance (FIG. 25). Furthermore,microarray data indicate activation of over half a dozen proto-oncogenesthat are slightly overexpressed in PASCs relative to ADSCs. The level ofexpression of these proto-oncogenes would be expressed at significantlylower levels (several orders of magnitude) in PASCs compared topluripotent embryonic stem cells and induced pluripotent stem cells(iPS) (23).

Conversion of PASCs into cancer stem cells (CSCs) can be induced ifPASCs are activated under carcinogenic stress conditions (e.g. ionizingradiation, ultraviolet light, chemical compounds, error-prone DNArepair, etc). Similarly, programming PASCs with Oct4 (Pou5f1), Sox2,cMyc, and Klf4 (Yamanaka's factors) lead to the formation of iPS (cellswith teratogenic potential) (FIG. 25).

Canonical pathways include the role of Oct4 in embryonic stem cellpluripotency, BRCA1 in DNA damage and hereditary breast cancersignaling, cell cycle control of chromosomal replication, DNA repair,arginine degradation, and embryonic stem cell differentiation intocardiac lineages. These data provide further insight into the role ofPASCs in DNA repair, cell cycle, oxidative stress, cancer cellregulation, as well as their intrinsic pluripotency (FIGS. 16-17).

This cell type, as isolated and activated by these techniques wouldallow for: (1) early detection and identification of CSCs for moreaccurate and earlier diagnosis; (2) a model for study of CSCs usingendogenous and non-manipulated cells, ideal for use in high-throughputapplications, and used to develop biomarkers for detection, as well asdrugs and chemotherapeutic approaches for treatments; (3) controllingthese cells, via understanding inherent activation and their role in ahealthy human stem cell niche, leading to new approaches to inducingthese cells' natural resting or senescent state. This resting stateprovides a pool of cells that can be used and activated for tissuerepair of damaged endodermal, ectodermal, and mesodermal cell lineages.Unlike iPC, these cells have active tumor suppressors, and precursorproto-oncogenes (rather than induced oncogenes) making them much moresuitable for safe use in regenerative applications, once properlydifferentiated.

Role of PASCs in Human Reproduction

A significant cause of infertility is reduced mitochondrialfunctionality in the oocytes of women undergoing in vitro fertilization(IVF) therapy. PASCs normally exist in a quiescent state and areactivated by severe cellular stress. PASCs are inherently resilient tothe growing environmental hostility of an advanced maternal age (AMA)ovary, in which oocyte mitochondria are highly susceptible to thedetrimental effects of hypoxia and apoptosis. PASCs could thereforecontribute to autologous mitochondrial transfer to oocytes of AMA womenwith infertility due to their intrinsic stress endurance and potentialto adopt the germ cell lineage. This would avoid the deleteriousheterogeneity that has arisen in previous trials with third-party andsomatic cell transfers.

PASCs can be isolated from adipose tissue from a young female withhealthy mitochondria, or from a sibling or mother with identicalmitochondria. This source of healthy mitochondria can be used as atreatment in one of two ways: (1) by transferring healthy PASCmitochondria to the oocyte that will be fertilized, or (2) bydifferentiating the healthy PASCs to an oocyte phenotype and swappingout the “donor” PASC nucleus (that has healthy mitochondria in thecytoplasm) with the nucleus of the oocyte with dysfunctionalmitochondria.

Furthermore, as inherent oocyte production in the mammalian femaleterminates at birth, excessive depletion of the existing pool offollicles due to reproductive abnormalities is essentially irreparablein vivo. Generation of human oocytes derived from PASCs offers a meansof autologous transplantation to provide functional oocytes for commoncauses of infertility, including premature ovarian failure, reproductiveaging associated with delayed childbearing, and poor oocyte quality,which can occur even in young women. In addition, the study of inducedgametogenesis and oocyte generation from PASCs can provide furtherinsight into the biological mechanisms of mammalian gametogenesis andthe female reproductive function in its entirety.

The foregoing methods are intended to be illustrative and not limiting.Using the teaching provided herein, other methods of isolating and usingPASCs will be available to one of skill in the art.

Examples

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1: Isolation and Characterization of PASCs

This example describes the isolation and characterization of PASCs. Insummary, the example shows the following: PASCs can grow in suspensionas individual cells and cell spheres reminiscent of embryonic stem cellclusters, and have the capacity for self renewal, yet grow at a moderaterate (FIG. 7) Karyotype studies indicate that PASCs have 23 pair ofchromosomes which show normal integrity (FIG. 8) A small percentage ofPASCs (10-20%) can be recognized by CD90 (marker for thymocytes), CD73(marker for lymphocyte differentiation), CD34 (marker for hematopoieticstem cells), CD45 (marker for hematopoietic stem cells), CD44 (markerfor activated T lymphocytes), and HLA-DR (marker for HLA class II) (FIG.9A-B). In contrast, 60-70% of PASCs were recognized by CD105, CD29(marker for T cells) and HLA-ABC (marker for HLA class I) (FIG. 9A-B).These results confirm that PASCs have the potential to be used in celltherapy for allotransplantation open the possibility of PASC celltransplantation between different individuals without immunorejection.Furthermore, these results clearly indicate the phenotypic differencesbetween PASCs and Muse cells, another pluripotent stem cell resistant tocellular stress and isolated by CD105/SSEA3 cell sorting.

In addition, PASCs are positive for the pluripotency markers SSEA3,TR-1-60, Oct3/4, Nanog and Sox2 (FIGS. 10A-B), and can spontaneouslydifferentiate into mesenchymal, endodermal and ectodermal cell lineageswith an efficiency between 20-23% (FIGS. 11A, 12A, 13A). In the presenceof specific differentiation media, PASCs can be induced to mesenchymal,endodermal and ectodermal with an efficiency between 78-82%. (FIGS.11B-C, 12B, 13B-C). While regular ASCs require 14-17 days of treatmentin adipogenic medium to become adipocytes, PASCs can be rapidlyconverted to adipocytes like cells by only 5 days of treatment inadipogenic medium (FIG. 11B). And while regular ASCs require 21 days oftreatment in myogenic medium to become myocytes, PASCs can be rapidlyconverted to myocyte like cells with only 5 days of treatment inmyogenic medium (FIG. 11C). PASCs do not undergo cell proliferation ortumor formation, setting them apart from ES and iPS cells. At 4 monthsafter transplantation into the testes of immune-deficient mice, PASCs(10⁶ cells/mouse) did not form teratomas. In contrast, the tumorigeniccell line F19 formed teratomas at 3 weeks-post injection (FIG. 14).MicroRNA Let-7 is the most significant upstream regulator present inPASCs vs ASCs (FIG. 18). Lin28, a RNA-binding protein gene, maintainsboth pluripotency and tumorigenesis in ES and iPS cells. Over-expressionof Let-7 in Muse cells would potentially play a critical role ininhibiting Lin28 expression, and therefore would protect these cellsfrom tumorigenic proliferation and teratoma formation.

PASCs have a very low level of cell division and proliferation, withouttumorigenic activity. In contrast, PASCs exhibit a very high level ofexpression of genes associated with immunity, inflammation, immuneresponse, immune suppression, lymphocyte activation and T cellactivation, suggesting a role for PASCs in dendritic cells or T-helpercells at the site of an infection (FIG. 15).

Microarray data indicate a substantial up-regulation of Sox2, Oct3/4,and Rex1 in PASCs relative to ADSCs. PASCs also exhibit gene expressionpatterns associated with the down-regulation of genes involved in celldeath and survival, embryonic development, DNA replication and repair,cell cycle and potential factors related to oncogenicity. Geneexpression analysis indicates that PASCs and ADSCs are mesenchymal inorigin; however, PASCs also express numerous lymphocytic andhematopoietic genes, such as CCR1 and CXCL2, encoding chemokinereceptors and ligands involved in stem cell homing. Gene ontologyanalysis of PASCs vs ASCs indicate that the most statisticallysignificant categories of cellular functions include cell death andsurvival, embryonic development, tissue development, cellular assemblyand organization, cellular function and maintenance, DNA replication,recombination and repair, cell cycle, organ development and organismalsurvival (FIG. 16). Canonical pathways provide further insight into thepotential role PASCs in DNA repair, cell cycle, oxidative stress, cancercell regulation, as well as their intrinsic pluripotency (FIG. 17). Inaddition, PASCs exhibit phagocytic activity. Unlike macrophages thatphagocytize entire cells, PASCs leave the nucleus of mature cellsuntouched, which could be used by PASCs as genetic information togenerate identical and healthy mature cells (FIG. 19)

Materials and Methods

Isolation of PASCs from Lipoaspirated Fat

Lipoaspirates (50 ml-2000 ml) were obtained from subcutaneous abdominaladipose of women undergoing liposuction. Lipoaspirate was repeatedlywashed with PBS until blood was completely removed from the tissue. 50ml-2000 ml of lipoaspirate was mixed with equal volume Dulbecco'sModified Eagle Medium 1× (DMEM, CellGro, MediatechInc, Manassas, Va.)containing collagenase (0.1%, Sigma Aldrich) in 50 ml tubes andincubated for 30 min at 37° C. in a shaking incubator at 110 rpm. Ifadipose tissue was not completely digested the incubation was continuedunder the same conditions for additional 10-15 more minutes, followed byincubation in 4° C., while still in collagenase and nutritionallydeficient medium (no FCS), for 6 hours. For this, the 50 ml tubes weretransferred to a regular 4° C. refrigerator allowing the temperature toslowly drop from 37° C. to 4° C. Under these severe cellular stressconditions (prolonged collagenase incubation, lack of nutrients, lowtemperatures and high hypoxia) all cells present in the lipoaspiratedigested material died, except PASCs highly resistant to such stress.Digested material was then centrifuged at 1500 rpm for 10 minutes at 4°C. Supernatant containing adipose cell debris (dead adipocytes,macrophages, red blood cells, adipose stem cells among other cellcomponents) was removed by aspiration and the remaining cell pelletswere combined and transferred to a new 50 ml tube. Cell pellet was thenwashed three times with 25 ml DMEM each time to assure that allcollagenase was completely removed from cell pellet. Cell werere-suspended in DMEM comprised of 10% fetal bovine serum (FBS; ThermoScientific Hyclone, Logan, Utah) and 5% antibiotic-antimycotic solution(CellGro, Mediatech Inc, Manassas, Va.), and plated as cells insuspension as well as adherent cells to further characterize the purityand pluripotency of PASCs. If PASCs will be injected in an animal model,freshly isolated PASCs will be resuspended in a small volume of salinesolution and directly injected into the damaged area or via i.v.following standard protocols.

Cell Expansion, Clonality and Karytotype of PASCs

Cell expansion and clonality of PASCs were determined after 8 differentpassages. Cell clusters spheres were formed after few hours of platingfreshly isolated PASCs in non-adherent dish (1^(st) generation). Once,most of cell spheres reached a size >50 μm, cell clusters weredisaggregated by pipetting and transfer to a new non-adherent dish(2^(nd) generation). PASCs were grown forming new cell-clusters asbefore with a growth rate of 1½ days/cell division (3^(rd) generation).The karyotypes of all expanded cells were determined byquinacrine-Hoeschst staining following standard protocols.

Flow Cytometry Analysis

Floating PASCs were cultured in DMEM/10% FCS for 8 hours followed byFACS analysis. Cells were washed in 2% inactivate FCS/0.05% sodiumAzide/PBS and were re-suspended in 100 μl of the same buffer andincubated at 4° C. for 1 hour in the presence or absence of primaryunconjugated rat anti-human SSEA3 (EMD Millipore; Billerica, Mass.).Cells were then washed twice with the same buffer and incubated with thecorresponding secondary FITC-conjugated anti-rat IgM (BD Biosciences;San Diego, Calif.) for 45 minutes at 4° C. Cells were then washed andre-suspended in 200 μl of the same buffer. Analysis of count and celltype was performed using a FACS Calibur flow cytometer and cEllQuest Prosoftware. For further characterize PASCs, other markers, such as CD105(marker of mesenchymal stem cells), CD90 (marker for thymocytes), CD73(marker for lymphocyte differentiation), CD34 (marker for hematopoieticstem cells), CD45 (marker for hematopoietic stem cells), CD44 (markerfor activated T lymphocytes), HLA-DR (marker for HLA class II) were usedfor FACS following standard protocols.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde (20 min at R/T), washed in PBS,then incubated in 0.2% Triton for 20 min. After 2 successive washes inPBS, cells were blocked with 10% normal goat serum in 1% BSA solutionfor 60 min at R/T. Cells were then incubated with the primary antibodiesovernight at 4° C. The following pluripotent stem cell markers wereused: rat anti-human stage-specific embryonic antigen (SSEA3, Millipore,Billerica, Mass.), mouse anti-human octamer-binding transcription factor3 and 4 (Oct3/4, Santa Cruz Biotech, Santa Cruz, Calif.), rabbitanti-human Nanog (Millipore, Billerica, Mass.), rabbit anti-humanSRY-box 2 (Sox2, Millipore, Billerica, Mass.), and mouse anti-humanTRA-1-60 (Abcam, Cambridge, Mass.); for mesenchymal cell lineages:rabbit anti-human preadipocyte factor 1 (Pref-1, [a.k.a. delta-like 1homolog (drosophila), DLK1] preadipocyte marker, Santa Cruz Biotech,Santa Cruz, Calif.); mouse anti-human myosin D (MyoD, myocyte marker,R&D Systems, Minneapolis, Minn.), and mouse anti-human smooth muscleactin (SMA, myocyte marker, Thermo Scientific, Waltham Mass.); forendodermal cell lineages: mouse anti-human pan keratin (Santa Cruz,Calif.); rabbit anti-human α-fetoprotein (Dako, Santa Clara, Calif.);and mouse anti-human cytokeratin 7 (Millipore, Billerica, Mass.); andfor ectodermal cell lineages: mouse anti-human neuron specific enolase(NSE, Millipore, Billerica, Mass.); rabbit anti-human glutamate receptor(Abcam, Cambridge, Mass.); rabbit anti-human NeuroD (Chemicon, TemeculaCalif.); mouse anti-human nestin (Chemicon, Temecula Calif.); and rabbitanti-human microtubule-associated protein 2 (MAP2, AbDSerotech, Raleigh,N.C.). All primary antibodies were diluted 1:200 in PBS/0.1% BSAsolution. Following treatment with primary antibodies, cells were washed3 times with PBS and incubated for 1 hour at R/T with PBS/0.1% BSAcontaining secondary immunofluorescent antibodies (1:1000) Alexa Fluor488 conjugated dye (mouse or rat, Invitrogen, Carlsbad, Calif.) or TexasRed conjugated dye (rabbit, Invitrogen, Carlsbad, Calif.). Cells werewashed 4× with PBS and treated with PBS/0.2% DAPI for 10 minutes. Cellswere then washed 3× with PBS. Images were acquired with an Evosimmunofluorescence inverted microscope (Advanced Microscopy, Mill Creek,Wash.).

Results

I— PASCs Isolation from Lipoaspirated Human Adipose Tissue

Adipose tissue is composed of adipocytes (mature cells) and the stromalvascular fraction (SVF) containing a heterogeneous population of cells,including adipose tissue macrophages (ATMs), adipose stem cells (ASCs),mesenchymal stem cells, and fibroblasts (24-25). Isolated PASCs wereactivated from their quiescent state by exposing them to cellular stress(FIG. 4-5).

For this, lipoaspirate material was first incubated in collagenase for30 min at 37° C. to release adipocytes (floating cells) and differentcellular components present in the SVF as previously described (25).This material was then subjected to severe cellular stress, includinglong incubation with collagenase, low temperatures, low serum andhypoxia all cells die under these conditions, except PASCs because oftheir high resistance to cellular stress.

Optimal conditions for the release of PASCs were determined to be 6-8hours incubation with collagenase in DMEM medium without FCS at 4° C.under very low 02, which subsequently gave way to a homogenouspopulation of PASCs. This high purity is presumably due to the severityof the cellular stress conditions, responsible for the depletion ofother cell types. As all other components of the adipose tissuelipoaspirate failed to survive, a population of highly purified PASCswas obtained, and therefore further purification processes were notnecessary.

PASCs were plated in both adherent and non-adherent cell culture dishes.It was observed that PASCs can grow either in suspension or in adherenceculture to form the characteristic cell clusters observed in EScell-derived embryoid body, as described in bone marrow and dermalfibroblast-derived Muse cells in previous reports (FIG. 5) (22-23).Under both conditions, individual PASCs reached a diameter of around 10μm and cell clusters reached a diameter of up to 50-100 μm by day 1without major changes in the number of isolated cells and cluster cells(FIG. 7), confirming previous data of the limited of proliferativecapacity of Muse cells (22).

II—PASCs have the Capacity of Self Renewal and Clonal Expansion

PASCs have the capacity for self renewal least for 8 different passages.Cell clusters spheres are formed after few hours of plating PASCs innon-adherent dish (1^(st) generation). Once, most of cell spheresreached a size >50 μm, cell clusters were disaggregated by pipetting andtransfer to a new non-adherent dish (2^(nd) generation). PASCs weregrown forming new cell-clusters as before with a growth rate of 1½days/cell division (3^(rd) generation). This indicates that PASCs have avery slow growth rate (FIG. 7)

III—PASCs have Normal Karyotype

The karyotypes of all expanded cells were normal without showing anychromosomal abnormalities. The 23 pair of chromosomes have shown normalintegrity including the sex chromosomes XX (female donor) (FIG. 8)

IV—Characterization of PASCs by Fluorocytometry Analysis

Specific antibodies for SSEA3, a cell-surface glycosphingolipidfrequently used to detect human ES cells and to purify Muse cells frombone marrow and dermis (61) and CD105, a classical marker of mesenchymalstem cells (43) was used for the FACS analysis. Approximately 40-50% ofPASCs were positive for both markers. Interestingly, Muse cells (anotherpluripotent stem cells resistant to cellular stress) isolated by cellsorting were 100% positive for both SSEA3 and CD105 antibodies (22-23).

10-20% of PASCs were recognized by CD90 (marker for thymocytes), CD73(marker for lymphocyte differentiation), CD34 (marker for hematopoieticstem cells), CD45 (marker for hematopoietic stem cells), CD44 (markerfor activated T lymphocytes), HLA-DR (marker for HLA class II). Incontrast, 40-60% of PASCs were recognized by CD105 (marker formesenchymal stem cells), 60-70% by CD29 (marker for T cells) and HLA-ABC(marker for HLA class I) (FIG. 9A-B). These results confirm that PASCshave the potential to be used in cell therapy for allotransplantation.Interestingly, Muse cells derived from bone marrow and skin cells havedifferent phenotype characteristics than PASCs. For example, Muse cellsare not recognized by CD34 and CD29 while all Muse cells express SSEA3,CD105 and CD90 (22). Muse cells are isolated by cell sorting, usingSSEA3/CD105 antibodies without the use of cellular stress treatment(22-23). These results indicate that PASCs and Muse cells have differentphenotypes and are therefore different cell types.

V—Characterization of PASCs by Immunocytochemistry

Upon transfer and adherence to chamber slides for immunofluorescentstaining, both the PASCs cell clusters and individual PASCs stronglyexpressed all of the characteristic pluripotent stem cell markers thatwere examined. These included SSEA3, a cell-surface glycosphingolipidfrequently used to detect human ES cells and to purify Muse cells frombone marrow and dermis (61, 22-23); Oct3/4 a protein involved in theself-renewal of human ES cells; Nanog, a transcription factor involvedin the self-renewal of human ES cells; Sox2, a transcription factor thatcontrols genes involved in embryonic development; and TRA-1-60, whichreacts with the antigen TRA-1-60 on the surface of embryonic germ cellsand ES cells (FIG. 10A-B). Comparatively, ADSCs derived from the samelipoaspirated tissue were either negative or weakly positive for thesepluripotent stem cell markers (FIG. 10A-B).

Example 2: Differentiation of PASCs into the Three Germline CellLineages Material and Methods Immunocytochemistry

Fixed PASCs were subjected to immunocytochemistry studies to determinetheir germline cell origin. For endodermal cell lineages: mouseanti-human pan keratin (Santa Cruz, Calif.); rabbit anti-humanα-fetoprotein (Dako, Santa Clara, Calif.); and mouse anti-humancytokeratin 7 (Millipore, Billerica, Mass.); and for ectodermal celllineages: mouse anti-human neuron specific enolase (NSE, Millipore,Billerica, Mass.); rabbit anti-human glutamate receptor (Abcam,Cambridge, Mass.); rabbit anti-human NeuroD (Chemicon, Temecula Calif.);mouse anti-human nestin (Chemicon, Temecula Calif.); and rabbitanti-human microtubule-associated protein 2 (MAP2, AbDSerotech, Raleigh,N.C.). All primary antibodies were diluted 1:200 in PBS/0.1% BSAsolution. Following treatment with primary antibodies, cells were washed3 times with PBS and incubated for 1 hour at R/T with PBS/0.1% BSAcontaining secondary immunofluorescent antibodies (1:1000) Alexa Fluor488 conjugated dye (mouse or rat, Invitrogen, Carlsbad, Calif.) or TexasRed conjugated dye (rabbit, Invitrogen, Carlsbad, Calif.). Cells werewashed 4× with PBS and treated with PBS/0.2% DAPI for 10 minutes. Cellswere then washed 3× with PBS. Images were acquired with an Evosimmunofluorescence inverted microscope (Advanced Microscopy, Mill Creek,Wash.).

Induced Differentiation of PASCs

Various differentiation media were used to induce differentiation ofMuse cells-AT to the three germline cell lineages.

For adipocyte formation, adherent PASCs were treated with adipogenicdifferentiation medium containing DMEM with 0.5 mMisobutylmethylxanthine, 1 μM dexamethasone, 10 μM insulin, 200 μMindomethacin and PPAR-γ (ZenBio, Inc, Research Triangle Park, N.C.) over3 or 6 days at 37° C. and 5% CO2. Adipocytes were detected usingfluorescence lipid drop marker BODIPY-C₁₆(1:1000, Invitrogen, Carslbad,Calif.) following manufacturer specification.

For myocyte formation, adherent PASCs were incubated in DMEM containingwith 10% FBS, 5% NHS, 50 μM hydrocortisone, and 1%antibiotic-antimycotic solution over 3 or 6 days at 37° C. and 5% CO2.Smooth muscle cells were identified by expression of smooth muscle actin(SMA) and skeletal muscle cells myosin D.

For hepatocyte and biliary cell induction, adherent PASCs were incubatedin hepatocyte differentiation medium for 3 or 6 days, as previouslydescribed adherent PASCs were incubated in DMEM supplemented with 10%FBS, 10 μg/ml insulin, 5.5 μg/ml transferring, 6.7 ng/ml sodium selenite(ITS; Gibco, Life Technologies, Grand Island, N.Y.), 10 nM dexamethasone(Sigma-Aldrich, St. Louis, Mo.), 100 ng/ml hepatocyte growth factor(HGF, Peprotech, Rocky Hill, N.J.) and 50 ng/ml and fibroblast growthfactor-4 (FGF-4, R & D Systems, Minneapolis, Minn.) (62). for 3 or 6days. Hepatocytes were identified by immunohistochemistry usingcytokeratin 7 and α-fetoprotein expression (see above).

For neural cell formation, Muse cells-AT were incubated as non-adherentcells in ultra-low attachment plates (Corning Incorporated, LifeSciences, Manassas, Va.) in the presence of neural differentiationmedium 1 containing Neurobasal medium (Gibco, Life Technology, GrandIsland, N.Y.) supplemented with B-27 supplement serum free (Gibco, LifeTechnology, Grand Island, N.Y.), 100 μg/ml kanamycin (Gibco, LifeTechnology, Grand Island, N.Y.), 2 mM glutamine (Sigma-Aldrich, St.Louis, Mo.), 30 ng/ml bFGF (Peprotech, Rocky Hill, N.J.) and 30 ng/mlEGF (Peprotech, Rocky Hill, N.J.) for 7 days (63). Cells were thentransferred to polystyrene culture slides (BD Biosciences, San Jose,Calif.) and cultured for another 7 days as adherent cells in thepresence of neural differentiation medium 2 containing 1 DMEMsupplemented with 2% FCS, 25 ng/ml bFGF and 25 ng/ml BDNF (Peprotech,Rocky Hill, N.J.). Neural cells were identified by immunohistochemistryusing nestin and MAP2 as indicated above.

Results I—Mesodermal Differentiation of PASCs

Adipose tissue has been shown to harbor ASCs with the ability todifferentiate into the mesenchymal cell lineages: adipocytes,chondrocytes, myocytes and osteoblasts [16, 20]. However, ASCs in theappropriate differentiation medium require approximately 2½ weeks todevelop mature adipocytes, and myocyte differentiation, withcharacteristically fused nuclei, takes approximately 3 weeks (24-25).

To determine the potential of PASCs to spontaneously differentiate intocells of mesodermal lineage, PASCs were grown as adherent cells inculture medium only containing DMEM, 10% FCS+Antibiotics for 3 days.Spontaneous differentiation of PASCs into a mesodermal lineage wasdetermined by immunocytochemistry. Mesodermal markers included DLK, amarker for preadipocytes (59), BODIPY-C₁₆, a fluorescent dye used todetect lipid accumulation (25, 64-65), and myosin D (Heavy Chain), amarker for the heavy chain portion of the Myosin II protein found inskeletal muscle cells (66, 67). After cultured as adherent cells for 3days, PASCs displayed significant expression of DLK, (21±8% of allDAPI-positive cells), BODIPY-C₁₆ (61±13% of all DAPI-positive cells) andMyosin D (25±4% of all DAPI-positive cells), as compared to ASCs, whichwere slightly positive only in response to DLK (17±7% of allDAPI-positive cells) (FIG. 11A).

In the presence of adipogenic medium, demonstrated over the course of 3and 6 days, PASCs accumulated considerable concentrations of lipid dropsindicated by the formation of BODIPY-C₁₆ (+) droplets, whichcharacterized 80±4% (3d) and 83±3% (6d) of all DAPI-positive cells (FIG.11B). In contrast, ASCs showed a weak yet detectable signal forBODIPY-C₁₆ (+) due to the presence of lipid accumulated in the cytoplasmas a result of ASC commitment to the preadipocyte cell fate (FIG. 11B).At 3 days that there is a stark morphological difference between PASCsand ASCs, perhaps most apparent in the smaller size of PASCs, which isvery evident in the nucleus size, as indicated by DAPI. However, thismorphology is actually much more similar at day 6, at which point thenucleus size has grown significantly in PASCs, and is roughly the sameas ASCs. BODIPY labeling of lipids with the intensity observed in thePASCs is typically observed in ASCs after 2-3 weeks in culture (25,6-65). Predictably, ADSCs fully differentiated to adipocytes after 17days of incubation in adipogenic medium (25).

In the presence of myogenic medium for 3 and 6 days, PASCsdifferentiated into smooth muscle cells, with the characteristicmorphology of smooth muscle fibers and strong expression of SMA thatcharacterized 77±3% (3d) and 83±4% (6d) of all DAPI-positive cells (FIG.11C). Under the same culture conditions, ADSCs were only slightlypositive after 6 days of incubation (25±4% of all DAPI-positive cells)(FIG. 10C).

Differentiation of ASCs to myocytes required ADSCs exposure to myogenicmedium for at least 21 days (data not shown, (25, 66-67). These resultsdemonstrate that while both types of adipose-derived stem/progenitorcells have the capacity to differentiate, activated PASCs differentiatetowards both adipocyte and myocyte lineages much more quickly than ASCs.

II—Endodermal Differentiation of PASCs

Spontaneous differentiation of PASCs to an endodermal lineage(hepatocytes) was detected in PASCs cultured in DMEM/10% FCS for 3 days.PASCs were recognized by α-fetoprotein (19±7% of all DAPI-positivecells), which is expressed during the development of endoderm andprogenitors of hepatocytes (68) and pan keratin (21±8% of allDAPI-positive cells), a marker for filaments characteristic of biliarytract epithelial cells [27] (FIG. 12A). In the cluster of cells,α-fetoprotein strongly recognized fatty acids in dimeric and trimericforms localized in both the cytoplasm and plasma membrane of PASCs (FIG.12A), as was previously described in human hepatoblastoma cell lineHepG2 (68). In contrast, ASCs were negative for these endodermal cellmarkers (FIG. 12A).

PASCs previously incubated in hepatogenic differentiation medium for 3and 6 days were positive for cytokeratin 7, an intermediate filamentprotein in biliary cells that characterized 69±2% (3d) and 80±7% (6d) ofall DAPI-positive cells, as well as for α-fetoprotein which recognized90±4% (3d) and 91±5% (6d) of all DAPI-positive cells (FIG. 12B), whileASCs were negative (FIG. 12B). These results demonstrate that PASCsdifferentiation mirrors previous studies of pluripotent stem cellsdifferentiation to hepatocytes in terms of both time in culture (3 days)and differentiation efficiency (4-5).

III—Ectodermal Differentiation of PASCs

PASCs were cultured for 3 days in DMEM/10% FCS with antibodiescharacterized by ectodermal cell markers including neuron-specificenolase (NSE), a marker used to detect neocortical neuron progenitors(69-70), metabrotopic-glutamate receptor (Glut-R), a marker used todetect microglial and neural like cells (71-72) and NeuroD, a markerused to detect neocortical precursor cells. Again, PASCs showedsignificant expression of all these markers with 30±5% (Glut-R), and15±5% (NeuroD) of all DAPI-positive cells (FIG. 13A), confirming theirpotential to spontaneously differentiate into ectodermal cells, asopposed to control ASCs (FIG. 13A).

The morphological progression of PASCs into neurons was monitoredthroughout the incubations in both the first and second neurogenicdifferential mediums (see Material and Methods) following similarprotocols previously used for the differentiation of ES and iPS cellsinto cells of neural origin (69-73). PASCs exhibited a progression fromthe formation of large cell spheres with finger-like projections tolong, neuron-like cells, which subsequently formed large networks (FIG.13B) (69). PASCs cultured in suspension for 7 days in neuraldifferentiation medium 1 (see Material and Methods) progressively formlarge cell clusters. Subsequent treatment as adherent cells for 7 daysin neural differentiation medium 2 ((see Material and Methods) inducedthe formation of neuron-like cells detectable by morphology and by axonand dendrite-specific markers.

Immunocytochemistry studies on PASCs having previously undergone thetwo-tiered neurogenic differentiation revealed expression of bothnestin, a stimulant of survival, renewal and proliferation of neuralprogenitor cells (74). which characterized 65±11% of all DAPI-positivecells and MAP2, a protein involved in the polymerization of microtubules[43] which recognized 92±2% of all DAPI-positive cells (FIG. 13C). ASCswere negative for nestin, while MAP2 exhibited a minimal degree ofnon-specificity inherent to the marker (5% of all DAPI-positive cells)(FIG. 13C).

Example 3: Gene Expression of PASCs Material and Methods MicroarrayAnalysis

PASCs and ASCs were isolated from lipoaspirate material of threedifferent patients. RNA was extracted using an RNeasy Mini Kit (Qiagen)and analyzed by Hokkaido System Science Co. Ltd. Array signals wereprocessed and normalized using the GeneSpring GX version 12.1.0 (AgilentTechnologies). Data has been deposited into the Gene Expression Omnibusdatabank with the access number GSE46353. The criteria for selectingdifferentially-expressed genes were preset as at least 2-fold differencein either direction plus statistical significance (P<0.05, unpaired ttest). Microarray analysis was performed using the software program IPAvia a license to Ingenuity (analysis(dot) ingenuity(dot)com) to identify(1) functional pathways (cell function, physiological function,diseases), canonical signaling pathways networks of related genesderived from genes changed in the analyzed comparisons and upstreamregulators. Further information regarding gene function was obtainedfrom the program GeneDecks V3 at genecards(dot)org [20]. Statisticalanalyses were carried out by Fischer's exact test (as performedautomatically by the software). In determining which genes are onlyexpressed in either PASCs or ASCs, all samples, having been performed intriplicate, had to display uniform detection (indicated with at least100 standard units) or absence (at most 30 standard units) along with aP-value <0.05.

Results

I—Gene Expression of PASCs in Comparison with ASCs

Tables S1-2 show PASCs cell gene expression in comparison with ASCsperformed by microarray in RNA obtained from three different patients.Differential expression of at least a 2-fold change between Muse—ATcells versus ASCs was observed in 435 up and 434 and down-regulatedgenes respectively (p<0.05, Tables S1-S2). The most predominantup-regulated genes of PASCs versus ASCs included CXCL2 (777.8 fold),ESCM2 (153.2 fold) DLL1 (147.4 fold), NR4A2 (139.2 fold), ADAMTS9 (115.3fold), BMX (91.5 fold), MYZAP (87.6 fold), ALDH1A2 (47.1 fold) and SOD2(41.4 fold), indicating that these genes were otherwise turned off orsuppressed in ASCs (Table S1). The most predominant down-regulated genesincluded AK5 (136.6 fold), GRE1142 (115.2 fold), CEP55 (93.6 fold),BUB1B (66.4 fold), CDCA3 (62.5 fold), NUF2 (54.8 fold) and DEPDC1 (52.7fold) (Table S2) (26). (Each of Tables S1-S6 referenced herein can befound in Reference 26.)

Tables S1-2 also show that many of the differentially expressed genes inPASCs are highly conserved, with homologues present in numerous smallorganisms (yeast, S. Cerevisiae, C. elegans, chlamydomonas, Tcalifornica, drosophila, etc.). This indicates the possibility that Musecells play a role in a highly conserved cellular mechanism related tocell survival in response to severe cellular stress (32-33).

Tables S3-4 show show genes only expressed in PASCs cell and not inASCs. Ninety-nine genes were expressed in all PASCs samples and absentin all ASC samples. Genes expressed only in PASCs included TNFSF14(p<0.0002), IL3RA (p<0.0007), CSF3 (p<0.0013), IL10RA (p<0.004), GATA2(p<0.005), and BMP7 (p<0.02) (Table S3). Interestingly, PASCs expressednumerous CD-markers that ASCs did not, while no CD-markers were uniqueto only the ASCs (Table S4).

Tables S5 show genes only expressed in ASCs and not in PASCs. There are41 genes expressed by ASCs that are absent in PASCs (Table S5). Thesegenes were largely related to mitosis and cell cycling, and includedESCO2 (p<0.0007), KIF20A (p<0.0009), CENPF (p<0.0023), NEK2 (p<0.0029),RAB3B (p<0.0031), and FGF5 (p<0.0068).

14 individual genes related to DNA repair were up-regulated in PASCs vsASCs (Table S6A). Additionally, eight ABC-cassette genes were morehighly expressed in PASCs. (Table S6B). Finally, in order to examinemethods of cell communication, the expression of gap junction relatedgenes was analyzed, and it was observed that PASCs expressed threeconnexin genes GJA4, GJB2, GJB4, as well as Clorf71 (CNST), whichencodes the recently described connexin recycling protein, consortin[44] (Table S6C).

FIG. 14 represents microarray analysis of functional group of genesexpressed in PASCs and not in ASCs and vice versa. PASCs have very lowexpression of genes related with mitosis, cell cycle, cellproliferation, cell adhesion, DNA repair, cell survival, ubiquitination,actin remodeling, metabolism and genes activated in iPS in comparisonwith ASCs. For the contrary, PASCs have very high level of expression ofgenes related with immunity, inflammation, immune regulation, immuneresponse, immune suppression, lymphocyte activation, T cell marker, Tcell proliferation, T cell activation, co-stimulation. PASCs mayregulates dendritic cells or T-helper cells at the site of an infection.

II—Gene Ontology Analysis

Gene ontology analysis was performed, and observed differentialexpression in PASCs correlated strongly to categories of cellularfunctions, the most statistically significant being: cell death andsurvival (p=2.04E-05 to 3.15E-02), embryonic development (p=5.92E-05 to3.15E-02), tissue development (p=5.92E-05 to 3.15E-02), cellularassembly and organization (p=1.07E-04 to 3.15E-02), cellular functionand maintenance (p=4.04E-04 to 3.15E-02), DNA replication, recombinationand repair (p=1E-0.3 to 3.15E-0.2), cell cycle (p=1.12E-0.3 to3.15E-0.2), organ development (p=1.54E-0.3 to 3.15E-0.2) and organismalsurvival (p=2.63E-0.3 to 2.63E-0.3) (FIG. 15, Tables S1-S2 of reference(26).

III—Canonical Pathways

The most highly expressed canonical pathways include the role of Oct4 inembryonic stem cell pluripotency (SOX2, NR6A1, BRCA1, ASH2L, POU5F1),BRCA1 in DNA damage and hereditary breast cancer signaling(POLRJ2/POLR2J3, FANCB, POLR2J, CDK6, RPA1, PIK3R2, RFC5, BIM BRCA1,RFC3), cell cycle control of chromosomal replication (MCM6, ORC3, CDK6,RPA1), DNA repair (RPA1, RFC5, RFC3), arginine degradation (ALDH4A1,OAT), and embryonic stem cell differentiation into cardiac lineages(SOX2, POU5DF1) (FIG. 17). These data provide further insight into thepotential role PASCs in DNA repair, cell cycle, oxidative stress, cancercell regulation, as well as their intrinsic pluripotency.

Up and down-regulation of critical genes involved in cell death andsurvival (e.g. SGK1 (up1.6×), MDH1, ATF2, HSPA8, PDL43, BRD1, CALM1,NR4A2, GATA2, CDK6, NUF2, CDK6, BRC1, BUB1B and CCXL2) could contributeto Muse-AT cell resistance to severe cellular stress exposure.

The BRC1 DNA damage and repair pathway] is down-regulated in PASCsversus ASCs, indicating the high capacity of PASCs to resist DNA damageas a result of severe cellular stress.

IV—Upstream Regulator Analysis

MicroRNA Let-7 is the most significant upstream regulator present inPASCs vs ASCs. Let-7 regulates 11 downstream genes associated withdecrease of cell cycle division (e.g. CDCA3, CDC16), celldifferentiation (DZIP1), cellular growth and proliferation (SSR1), DNAreplication (MCM6), replication factor and cancer (RFC3, RFC5) and celldeath and survival (NUF2, BRCA1, BUB1B, CDC16) (FIG. 18).

Lin28, a RNA-binding protein gene, maintains both pluripotency andtumorigenesis in ES and iPS cells. Let-7, a microRNA that regulatesembryonic development, cell differentiation and tumor suppression, havethe opposite effect (75). While over-expression of Let-7 blocks Lin28gene expression, strong Lin28 expression degrades Let-7, maintaining abalance between the two reciprocally counteractive genes (75).

ES and iPS cells have a very high Lin28/Let7 ratio, which has beenthought to play a major role in their tumorigenic propensities (75). Inthe absence of a strong Lin28 influence, Muse cells retain theirpluripotent capacity (75). Over-expression of Let-7 in Muse cells wouldpotentially play a critical role in inhibiting Lin28 expression, andtherefore would protect these cells from tumorigenic proliferation andteratoma formation.

Example 4: Role of PASCs in Immune and Autoimmune Diseases Material andMethods

Formation of PASCs after Co-Culture Between Adipocyte and the StromalVascular Fraction

Human adipose tissue was finely minced and treated with collagenase for60 minutes at 37° C., in the transport buffer. The cell suspension wasthen filtered through a pre-moistened 150-micron nylon mesh (Small PartsInc., Miami Lakes, Fla.) and centrifuged for 2 min at 50×g at RT. Theupper phase (floating adipocytes) was separated from lower phase (SVF).Adipocyte fraction was washed twice and diluted in adipocyte culturemedium (DMEM, 1% BSA, 3% FCS, 100 U/ml Penicillin, 100 μg/mlStreptomycin). The lower phase was subjected to centrifugation for 5minutes at 500×g. The cell pellet (SVF) was resuspended in PBS andsubjected a Ficoll density gradient to further purify the SVF. Theinterface containing the SVF fraction was removed and washed with 5 mlof PBS at RT. After a final centrifugation for 5 minutes at 500×g, thecell pellet was resuspended in culture medium (RPMI medium supplementedwith 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mML-glutamine, 1% NEAA, 1% sodium pyruvate, and 10 ng/ml GM-CSF). Cellsfrom the SVF fraction (10⁶/ml) and adipocyte fraction (10⁶/ml) wereallowed to equilibrate separately overnight in their respective cellculture media. Coverslips were placed in each well forimmunofluorescence studies. Twenty-four hours later, resuspendedadipocyte fraction was added to the wells containing the SVF fraction atan approximate 1:1 ratio. The pooled cells were co-cultured for 2-24hours at 37° C. in 5% CO₂. At the end of the incubation period theadipocyte fraction was resuspended and transferred by pipette and placedin another well containing 1 ml of adipocyte culture medium (see above).The remaining SFV fraction was washed five times with 1 ml PBS, andcultured for an additional two days in fresh regular medium (see above).Coverslips were then collected for immunofluorescence (see below).Adipocyte and SVF fraction were also separately cultured under the sameconditions described above. Immunohistochemistry was performed usingstandard protocols and different markers, S-100 and DLK (Santa Cruz,Calif.), markers for preadipocytes; CD34 marker for hematopoietic stemcells and early formation of adipose stem cells (Zymed, San Francisco,Calif.) and different pluripoten stem cell markers (Nanog, SOX2, Oct3/4,see above) were used.

Effect of PASCs on Splenocytes Contain Specific CD4 (+) T Cell Epitopefor Diabetes Type I

Splenocytes of a NOD BDC2.5 transgenic mice were isolated using standardconditions. These splenocytes contain CD4 (+) T cells, which onlyrecognize a specific peptide named Mimetope (Mim) whose sequence is partof chromogranin A (ChgA) (76). Splenocytes (5×10⁵ cells) were culturedin 24-well clusters with Mimetope for 72 hours in the presence(co-culture) or absence of PASCs (10³-10⁵ cells/well). The number ofsplenocytes (cell proliferation) were determined by FACS on stainedcarboxyfluorescein-diacetate succinimidyl ester (CFSE) splenocytesbefore and after incubation with Mim±PASCs. IFNγ production wasdetermined by FACS and RIA respectively.

Effect of PASC Conditioned Medium on Splenocytes Contain Specific CD4(+) T Cell Epitope for Diabetes Type I

10⁶ PASCs were cultured in non-adherent dishes in DMEM/10% FCS.Conditioned medium was collected after 48 hours. Splenocytes (5×10⁵cells) isolated from NOD BDC2.5 transgenic mice were cultured in 24 wellclusters with Mimetope for 72 hours in the presence or absence of PASCconditioned medium at different dilutions (½ to 1/500 dilutions). Forsplenocytes quantification, CFSE labeled splenocytes were incubated withMim±PASC conditioned medium. CFSE dilution was analyzed by FACS todetermine cell proliferation. IFNγ production was determined by RIA.

Effects of PASCs in Mice Diabetic Models

(A) NODscid mice were made diabetic by multiple (45 mg/kg, fourconsecutive days) injections of streptozotocin (STZ). PASCs wereinjected (10⁶ cells, i.p.) into diabetic (glycemia >240 mg/dl) NODscidmice once or twice (as indicated). Glycemia and body weight weredetermined at time zero as well as at different times post injectionusing standard conditions. (B) Diabetic NOD mice are a strain of mice inwhich spontaneous diabetes occurs. Soon after onset of hyperglycemia inthe NOD mice, PASCs were injected (10⁶ cells, i.p.). Glycemia and bodyweight were determined at time zero as well as at different times postinjection using standard conditions. Some animals received a 2^(nd)injection of PASCs (10⁶ cells, i.p.)

Results I—Phagocytic Activity of PASCs

Formation of PASCs were detected after co-culture of adipocyte fraction(floating cells) and the stromal vascular fraction (adherent cells)(59). There is a clear interaction between preadipocytes (S-100 (+)cells) and PASCs (SSEA3(+) cells, red). (FIG. 18A) PASCs are firmlyattached to approximately a third of the preadipocytes. This attachmentis apparently selective since seemingly identical preadipocyte cells arecompletely engulfed in PASCs. (FIG. 18B) shows a pre-adipocyteundergoing mitosis left undisturbed by PASCs while the preadipocytedirectly above is engulfed by PASCs leaving only the DAPI positivenucleus uncovered. While both preadipocytes and PASCs are S-100+ andSSEA3, (FIG. 18C-a, C-b) indicate that pre-adipocytes would showpositive S-100 staining with fluorescent light exposure of 1-2 seconds,with PASCs would display positive results with only a 1 millisecondexposure time. The great discrepancy in exposure strengths suggests thatthe PASCs express S-100 at much higher levels, or more readily allowsS-100 anti-body bind to its domain. Individual PASCs were originallyidentified through the clear DAPI staining of their small nuclei. Therephagocytosis of preadipocytes by the much smaller PASCs. Thepreadipocytes could clearly be seen at different stages of beingdevoured. Unlike macrophages that phagocytize entire cells, PASCs leavethe nucleus of preadipocytes untouched, (FIG. 18A) often times resultingin preadipocyte nuclei that are left bare in culture (FIG. 18B).

II—PASCs as Antigen-Specific Immunomodulation Cells

PASCs significantly reduced the number of BDC2.5 splenocytes stimulatedby Mimetope in a dose-response manner. 10³-10⁴ PASCs produced maximuminhibition in cell proliferation (FIG. 20A-D). The effect of PASCs onINFγ secretion by BDC2.5 splenocytes stimulated by Mimetope was moredramatic. 10³ PASCs completely inhibited INFγ levels (FIG. 20E).

PASC conditioned media significantly reduced the secretion of INFγ byAg-specific stimulated splenocytes. PASC-CM #1 and #2 obtained fromPASCs isolated from two different patients showed similar results at ⅕dilution (FIG. 21A-B). However, T cell proliferation was not affected byboth PASC conditioned media (FIG. 21C-D).

II—Effects of PASCs in Mice Diabetic Models

The effects of PASCs were first analyzed in a NODscid mice in whichdiabetes was induced by multiple injections of streptozotocin. Alldiabetic NODscid mice receiving PBS (control group) had glycemia ≥500mg/dl by day 5 and, died or were moribund at day 6 after first injection(FIG. 22C). In contrast, most of PASCs-treated mice effects (10⁶ cells,i.p.) survived for prolonged period maintaining an increase in bodyweight gain almost until 6 weeks (FIG. 22A). Six out of seven micesurvived 8 weeks-post transplant and five out of seven, reached week-11post-treatment (FIG. 22A). Most of the PASCs treated mice maintainedoscillating glycemia below 500 mg/dl at least during 6-weekspost-treatment (FIG. 22B). These results clearly indicate that PASCsdramatically improved the loss of weight and glucose levels at leastafter 6 weeks of treatment. It is likely that these effects are relatedwith the capacity of PASCs to regenerate new pancreatic and kidney cellsin the diabetic animal model.

The effects of PASCs were also analyzed in naturally occurring diabetesNOD mice. Soon after onset of hyperglycemia, NOD mice reached bloodglucose levels ≥500 mg/dl (day 4) with constant lost of body weight. Atday 10, NOD diabetic mice died (n=2) (FIG. 23C). Contrary, three out offour Muse cells-injected NOD diabetic mice showed an increase in bodyweight gain until 5-weeks post-treatment (FIG. 23A). Blood glucoseshowed oscillating levels with a marked delay to reach ≥500 mg/dl duringa prolonged period (FIG. 23B). Undoubtedly, the transplanted diabeticmice had higher survival than controls. These results clearly indicatethat PASCs dramatically improved the loss of weight and glucose levelsat least after 6 weeks of treatment. It is likely that these effects arerelated with the capacity of PASCs to regenerate new pancreatic andkidney cells in the NOD mice.

REFERENCES

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-20. (canceled)
 21. A method of treating traumatic injury ordisease-associated damage in a subject, the method comprisingadministering a composition comprising pluripotent stem cells isolatedfrom adipose tissue (PASCs) to the subject under conditions permittingthe PASCs of the composition to divide and populate a site of tissuedamage, wherein the PASCs are isolated from adipose tissue by a methodcomprising: (a) releasing adipose cells, including an adipocyte fractionand a stromal vascular fraction, from an adipose tissue sample using aproteolytic enzyme that breaks the peptide bonds in collagen; (b)co-incubating the released adipocytes and the released stromal vascularfraction for 2-36 hours, wherein 4-24 hours of said co-incubation takesplace under stress conditions in a medium containing a proteolyticenzyme, in the absence of nutrients, under hypoxic conditions, anddecreasing the temperature to 4° C.; (c) recovering the viable cellsfollowing the co-incubation in step (b) by centrifuging to produce acell pellet, removing supernatant containing adipocyte cell debris byaspiration, washing the cell pellet to remove the proteolytic enzyme,and resuspending the recovered cells in media.
 22. The method of claim21, further comprising culturing the recovered cells for up to 48 hours.23. The method of claim 21, wherein the disease-associated damagecomprises damage associated with diabetes, vascular disease, infection,degenerative neurological disease, cancer, or autoimmune disease. 24.The method of claim 21, wherein the traumatic injury comprises hypoxia,bone injury, laceration, gunshot wound, or stroke.
 25. The method ofclaim 21, wherein the degenerative neurological disease comprisesParkinson's Disease, Alzheimer's Disease, or Huntington's Disease. 26.The method of claim 21, wherein the degenerative neurological disease isParkinson's Disease.
 27. The method of claim 21, wherein thedisease-associated damage comprises damage associated with infection.28. The method of claim 21, wherein the disease-associated damagecomprises damage associated with ageing.
 29. The method of claim 21,wherein the adipose tissue sample of step (a) is obtained vialipoaspiration.
 30. The method of claim 29, wherein the lipoaspirationis performed on the subject to whom the composition is administered. 31.The method of claim 29, wherein the composition is administered to thesubject within 6 hours of the lipoaspiration.
 32. The method of claim21, wherein the composition is administered intravenously.
 33. Themethod of claim 21, wherein the adipose tissue is allogeneic to thesubject.
 34. The method of claim 21, wherein the adipose tissue isautologous to the subject.
 35. The method of claim 21, wherein thecomposition comprises 20,000,000-200,000,000 PASCs.
 36. The method ofclaim 21, wherein the composition is at least 95% pure PASCs.
 37. Themethod of claim 21, wherein the PASCs have been frozen and thawed priorto administration to the subject.
 38. A method of treating Parkinson'sdisease in a subject, the method comprising administering a compositioncomprising pluripotent stem cells (PASCs) to the subject underconditions permitting the PASCs of the composition to divide andpopulate a site of tissue damage, wherein the PASCs are isolated fromadipose tissue by a method comprising: (a) releasing adipose cells,including an adipocyte fraction and a stromal vascular fraction, from anadipose tissue sample using a proteolytic enzyme that breaks the peptidebonds in collagen; (b) co-incubating the released adipocytes and thereleased stromal vascular fraction for 2-36 hours, wherein 4-24 hours ofsaid co-incubation takes place under stress conditions in a mediumcontaining a proteolytic enzyme, in the absence of nutrients, underhypoxic conditions, and decreasing the temperature to 4° C.; (c)recovering the viable cells following the co-incubation in step (b) bycentrifuging to produce a cell pellet, removing supernatant containingadipocyte cell debris by aspiration, washing the cell pellet to removethe proteolytic enzyme, and resuspending the recovered cells in media.39. The method of claim 38, further comprising culturing the recoveredcells for up to 48 hours.